US5681562A - Lymphokine gene therapy of cancer - Google Patents

Lymphokine gene therapy of cancer Download PDF

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US5681562A
US5681562A US08/352,990 US35299094A US5681562A US 5681562 A US5681562 A US 5681562A US 35299094 A US35299094 A US 35299094A US 5681562 A US5681562 A US 5681562A
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tumor
cells
fibroblasts
cytokine
patient
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Robert E. Sobol
Fred H. Gage
Ivor Royston
Theodore Friedman
Habib Fakhrai
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Sidney Kimmel Cancer Center
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464499—Undefined tumor antigens, e.g. tumor lysate or antigens targeted by cells isolated from tumor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • lymphokines a subset of the cytokines. These agents mediate many of the immune responses involved in anti-tumor immunity.
  • cytokines Several of these cytokines have been produced by recombinant DNA methodology and evaluated for their anti-tumor effects.
  • the administration of lymphokines and related immunomodulators has resulted in objective tumor responses in patients with various types of neoplasms (4-7).
  • current modes of cytokine administration are frequently associated with toxicities that limit the therapeutic value of these agents.
  • interleukin-2 is an important lymphokine in the generation of anti-tumor immunity (4).
  • helper T-cells secrete small quantities of IL-2.
  • This IL-2 acts locally at the site of tumor antigen stimulation to activate cytotoxic T-cells and natural killer cells which mediate systemic tumor cell destruction.
  • Intravenous, intralymphatic and intralesional administration of IL-2 has resulted in clinically significant responses in some cancer patients (4-6).
  • severe toxicities hyperotension and adema
  • limit the dose and efficacy of intravenous and intralymphatic IL-2 administration (5-7).
  • the toxicity of systemically administered lymphokines is not surprising as these agents mediate local cellular interactions and they are normally secreted in only very small quantities.
  • cytokines such as interleukin-4 (IL-4), alpha interferon ( ⁇ -INF) and gamma interferon (c-INF) have been used to stimulate immune responses to tumor cells.
  • IL-4 interleukin-4
  • ⁇ -INF alpha interferon
  • c-INF gamma interferon
  • the present invention demonstrates a novel, more practical method of cytokine cancer immunotherapy.
  • selected cells from a patient such as fibroblasts, obtained, for example, from a routine skin biopsy, are genetically modified to express one or more cytokines.
  • patient cells which may normally serve as antigen presenting cells in the immune system such as macrophages, monocytes, and lymphocytes may also be genetically modified to express one or more cytokines.
  • cytokine-expressing cells ore CE cells.
  • the CE cells are then mixed with the patient's tumor antigens, for example in the form of irradiated tumor cells, or alternatively in the form of purified natural or recombinant tumor antigen, and employed in immunizations, for example subcutaneously, to induce systemic anti-tumor immunity.
  • tumor antigens for example in the form of irradiated tumor cells, or alternatively in the form of purified natural or recombinant tumor antigen, and employed in immunizations, for example subcutaneously, to induce systemic anti-tumor immunity.
  • the cytokines are locally expressed at levels sufficient to induce or augment systemic anti-tumor immune responses via local immunization at sites other than active tumor sites.
  • Systemic toxicity related to cytokine administration should not occur because the levels of cytokine secreted by the CE cells should not significantly affect systemic cytokine concentrations.
  • this approach provides the benefit of local cytokine administration.
  • this novel method obviates the need for intralesional injections, which may produce morbidity.
  • the continuous local expression of cytokine(s) at the sites of immunization may also augment anti-tumor immune responses compared to intermittent cytokine injections.
  • This method also provides the advantage of local immunization with the CE cells, as opposed to cumbersome intravenous infusions. This method also eliminates the need for establishing tumor cell lines in vitro as well as transfer of genes into these tumor cells.
  • This invention also provides an alternative means of localized expression of cytokines to induce and/or increase immune responses to a patient's tumor through genetic modification of cellular expression of both cytokine(s) and tumor antigen(s).
  • selected cells from a patient are isolated and transduced with cytokine gene(s) as well as gene(s) coding for tumor antigen(s).
  • the transduced cells are called "carrier cells.”
  • Carrier cells can include fibroblasts and cells which may normally serve as antigen presenting cells in the immune system such as macrophages, monocytes, and lymphocytes.
  • Transduced carrier cells actively expressing both the cytokine(s) and the tumor antigen(s) are selected and utilized in local immunizations at a site other than active tumor sites to induce anti-tumor immune responses.
  • these carrier cells should not produce substantial systemic toxicities, as the levels of cytokine(s) secreted by the carrier cells should not significantly affect systemic cytokine concentrations.
  • This alternate embodiment is advantageous because it obviates the need to obtain samples of the tumor, which is sometimes difficult.
  • carrier cells can be utilized in local immunizations in conjunction with tumor cells, tumor cell homogenates, purified tumor antigens, or recombinant tumor antigens to enhance anti-tumor immunity.
  • this second embodiment retains the same advantages as the first embodiment in that the level of cytokine released by the carrier cells is sufficient to induce anti-tumor immunity but is too low to produce substantial systemic toxicity.
  • this method obviates the need for intralesional injections, and allows for continuous expression of cytokine(s). This method also eliminates the need for establishing continuous cultures in vitro of tumor cells as well as transfer of genes into these tumor cells, and provides the advantage of local immunization with the carrier cells, as opposed to cumbersome lengthy intravenous infusions.
  • FIG. 1 shows schematic diagrams of retroviral vectors DC/TKIL2, LXSN-IL2, and LNCX-IL2.
  • FIG. 2 shows a mean IL-2 concentration of triplicate supernatant samples measured by ELISA. Supernatants were harvested from overnight cultures of approximately 1.5 ⁇ 10 6 semi-confluent fibroblasts.
  • FIG. 3 shows biological activity of the IL-2 secreted by the transduced fibroblasts was demonstrated by measuring mean 3 H-TdR incorporation of an IL-2 dependent T-cell line incubated with triplicate samples of supernatants. Supernatants were harvested from overnight cultures of approximately 1.5 ⁇ 10 6 semi-confluent fibroblasts.
  • FIG. 4 shows comparisons between animals injected with 10 5 CT26 tumor cells alone ( ⁇ ); 10 5 CT26 tumor cells and 2 ⁇ 10 6 unmodified BALB/C fibroblasts ( ⁇ ); 10 5 CT26 tumor cells and 2 ⁇ 10 6 IL-2 transduced BALB/C fibroblasts ( ⁇ ); and 10 5 CT26 tumor cells and 1 ⁇ 10 6 transduced BALB/C fibroblasts ( ⁇ ).
  • Tumor measurements are the mean products of the cross-sectional diameter of the tumors from four animals in each treatment group. The (*) indicates statistically significant difference (P ⁇ 0.05) in tumor growth curves.
  • FIG. 5 shows PCR analysis of neomycin phosphotransferase DNA sequences.
  • FIG. 6 shows the effect of IL-2 modified fibroblasts on tumor establishment and development using 2 ⁇ 10 6 fibroblasts mixed with 5 ⁇ 10 4 CT26 tumor cells concentrating on the rate of tumor growth.
  • FIG. 7 shows the effect of IL-2 modified fibroblasts on tumor establishment and development using 2 ⁇ 10 6 fibroblasts mixed with 5 ⁇ 10 4 CT26 tumor cells concentrating on the time of tumor onset for the individual animal in each treatment group.
  • FIG. 8 shows the effect of IL-2 modified fibroblasts on tumor establishment and development using 2 ⁇ 10 6 fibroblasts mixed with 1 ⁇ 10 5 CT26 tumor cells concentrating on the rate of tumor growth.
  • FIG. 9 shows the effect of IL-2 modified fibroblasts on tumor establishment and development using 2 ⁇ 10 6 fibroblasts mixed with 1 ⁇ 10 5 CT26 tumor cells concentrating on the time of tumor onset for the individual animal in each treatment group.
  • FIG. 10 shows the effect of IL-2 modified cells on tumor establishment and development using 2 ⁇ 10 6 DCTK-IL2-modified CT26 tumor cells mixed with 1 ⁇ 10 5 unmodified CT26 compared to 2 ⁇ 10 6 DCTK-IL2-modified fibroblasts mixed with 1 ⁇ 10 5 CT26 concentrating on the rate of tumor growth.
  • FIG. 11 shows the effect of IL-2 modified cells on tumor establishment and development using 2 ⁇ 10 6 DCTK-IL2-modified CT26 tumor cells mixed with 1 ⁇ 10 5 unmodified CT26 compared to 2 ⁇ 10 6 DCTK-IL2-modified fibroblasts mixed with 1 ⁇ 10 5 CT26 concentrating on the time of tumor onset for the individual animal in each treatment group.
  • FIG. 12 shows the effect of IL-2 modified fibroblasts on induction of systemic anti-tumor immunity and the rate of tumor growth.
  • Mice were immunized with 2 ⁇ 10 6 fibroblasts mixed with 2.5 ⁇ 10 5 irradiated CT26 tumor cells 7 days prior to challenge with 5 ⁇ 10 4 fresh tumor cells.
  • FIG. 13 shows the effect of IL-2 modified fibroblasts on induction of systemic anti-tumor immunity and the time of tumor onset for the individual animal in each treatment group.
  • Mice were immunized with 2 ⁇ 10 6 fibroblasts mixed with 2.5 ⁇ 10 5 irradiated CT26 tumor cells 7 days prior to challenge with 5 ⁇ 10 4 fresh tumor cells.
  • FIG. 14 shows the effect of IL-2 modified fibroblasts on induction of systemic anti-tumor immunity and the rate of tumor growth.
  • Mice were immunized with 2 ⁇ 10 6 fibroblasts mixed with 2.5 ⁇ 10 5 irradiated CT26 tumor cells 14 days prior to challenge with 5 ⁇ 10 4 fresh tumor cells.
  • FIG. 15 shows the effect of IL-2 modified fibroblasts on induction of systemic anti-tumor immunity and the time of tumor onset for the individual animal in each treatment group.
  • Mice were immunized with 2 ⁇ 10 6 fibroblasts mixed with 2.5 ⁇ 10 5 irradiated CT26 tumor cells 14 days prior to challenge with 5 ⁇ 10 4 fresh tumor cells.
  • a novel method of tumor immunotherapy comprising the genetic modification of cells resulting in the secretion of cytokine gene products to stimulate a patient's immune response to tumor antigens.
  • Gene is defined herein to be a nucleotide sequence encoding the desired protein.
  • autologous fibroblasts genetically modified to secrete at least one cytokine gene product are utilized to immunize the patient in a formulation with tumor antigens at a site other than an active tumor site.
  • cells genetically modified to express at least one tumor antigen gene product and to secrete at least one cytokine gene product are utilized in formulation to immunize the patient at a site other than an active tumor site.
  • Cytokines are preferably expressed in cells which efficiently secrete these proteins into the surrounding milieu.
  • Fibroblasts are an example of such cells.
  • Fibroblasts or other cells can be genetically modified to express and secrete one or more cytokines, as described later in this specification.
  • Tumor antigens can be provided by several methods, including, but not limited to the following: 1) CE cells can be transduced with gene(s) coding for tumor antigens. These "carrier cells” are then utilized in patient immunizations. 2) Cloned gene sequences coding for appropriate tumor antigens can be transferred into cells such as fibroblasts or antigen-presenting cells. These cells are then mixed with CE or carrier cells to immunize the patient. 3) Tumor antigens can be cloned in bacteria or other types of cells by recombinant procudures. These antigens are then purified and employed an immunization with CE and/or carrier cells.
  • Tumor antigens can be purified from tumor cells and used, along with CE or carrier cells, to immunize the patient.
  • Tumor cells may be irradiated or mechanically disrupted and mixed with CE and/or carrier cells for patient immunizations.
  • This invention encompasses the following steps: (A) isolation of appropriate cells for generation of CE cells or carrier cells; (B) isolation of cytokine genes or isolation of cytokine genes and tumor antigen genes, as well as appropriate marker and/or suicide genes; (C) transfer of the genes from (B) to produce the CE cells or carrier cells; (D) preparation of immunological samples of the patient's tumor antigens or other suitable tumor antigens for immunization with CE or carrier cells; (E) inactivation of the malignant potential of tumor cells if they are used as a source of tumor antigens for immunization; and (F) preparation of samples for immunization. Following are several embodiments contemplated by the inventors. However, it is understood that any means known by those in the art to accomplish these steps will be usable in this invention.
  • Cells to be utilized as CE cells and carrier cells can be selected from a variety of locations in the patient's body.
  • skin punch biopsies provide a readily available source of fibroblasts for use in generating CE cells, with a minimal amount of intrusion to the patient.
  • these fibroblasts can be obtained from the tumor sample itself.
  • Cells of hematopoietic origin may be obtained by venipuncture, bone marrow aspiration, lymph node biopsies, or from tumor samples.
  • Other appropriate cells for the generation of CE or carrier cells can be isolated by means known in the art. Non-autologous cells similarly selected and processed can also be used.
  • cytokine genes Numerous cytokine genes have been cloned and are available for use in this protocol. The genes for IL-2, c-INF and other cytokines are readily available (1-5, 11-14). Cloned genes of the appropriate tumor antigens are isolated according to means known in the art.
  • Selectable marker genes such as neomycin resistance (Neo R ) are readily available. Incorporation of a selectable marker gene(s) allows for the selection of cells that have successfully received and express the desired genes. Other selectable markers known to those in the art of gene transfer may also be utilized to generate CE cells or carrier cells expressing the desired transgenes.
  • “Suicide” genes can be incorporated into the CE cells or carrier cells to allow for selective inducible killing after stimulation of the immune response.
  • a gene such as the herpes simplex virus thymidine kinase gene (TK) can be used to create an inducible destruction of the CE cells or carrier cells.
  • TK herpes simplex virus thymidine kinase gene
  • a drug such as acyclovir or gancyclovir can be administered. Either of these drugs will selectively kill cells expressing TK, thus eliminating the implanted transduced cells.
  • a suicide gene may be a gene coding for a non-secreted cytotoxic polypeptide attached to an inducible promoter. When destruction of the CE or carrier cells is desired, the appropriate inducer of the promoter is administered so that the suicide gene is induced to produce cytotoxic polypeptide which subsequently kills the CE or carrier cell. However, destruction of the CE or carrier cells may not be required.
  • Genes coding for tumor antigen(s) of interest can be cloned by recombinant methods.
  • the coding sequence of an antigen expressed by multiple tumors may be utilized for many individual patients.
  • genes into cultured cells Numerous methods are available for transferring genes into cultured cells (15). For example, the appropriate genes can be inserted into vectors such as plasmids or retroviruses and transferred into the cells. Electroporation, lipofection and a variety of other methods are known in the field and can be implemented.
  • TILs tumor infiltrating lymphocytes
  • Neo R Neomycin resistance
  • replication competent retroviruses When retroviruses are used for gene transfer, replication competent retroviruses may theoretically develop by recombination between the retroviral vector and viral gene sequences in the packaging cell line utilized to produce the retroviral vector.
  • packaging cell lines in which the production of replication competent virus by recombination has been reduced or eliminated.
  • all retroviral vector supernatants used to infect patient cells will be screened for replication competent virus by standard assays such as PCR and reverse transcriptase assays (16).
  • exposure to replication competent virus may not be harmful.
  • the retrovirus was cleared by the primate immune system (17). No clinical illnesses or sequelae resulting from replication competent virus have been observed three years after exposure. In summary, it is not expected that patients will be exposed to replication competent murine retrovirus and it appears that such exposure may not be deleterious (17).
  • Tumor cells bearing tumor associated antigens are isolated from the patient. These cells can derive either from solid tumors or from leukemic tumors. For solid tumors, single-cell suspensions can be made by mechanical separation and washing of biopsy tissue (18).
  • Hematopoietic tumors may be isolated from peripheral blood or bone marrow by standard methods (19).
  • a second variant is the use of homogenates of tumor cells.
  • Such homogenates would contain tumor antigens available for recognition by the patient's immune system upon stimulation by this invention.
  • purified tumor antigens obtained for example by immunoprecipitation or recombinant DNA methods, could be used. Purified antigens would then be utilized for immunizations together with the CE cells and/or carrier cells described above to induce or enhance the patient's immune response to these antigens.
  • tumor antigens are available through their expression by the carrier cells. These carrier cells can be injected alone or in conjunction with other tumor antigen preparations or CE cells. Likewise, when CE cells are used, purified recombinant tumor antigen, produced by methods known in the art, can be used.
  • autologous tumor cells are not readily available, heterologous tumor cells, their homogenates, their purified antigens, or carrier cells expressing such antigens could be used.
  • the tumor cells When viable tumor cells are utilized in immunizations as a source of tumor antigens, the tumor cells can be inactivated so that they do not grow in the patient. Inactivation can be accomplished by several methods. The cells can be irradiated prior to immunization (18). This irradiation will be at a level which will prevent their replication. Such viable calls can then present their tumor antigens to the patient's immune system, but cannot multiply to create new tumors.
  • tumor cells that can be cultured may be transduced with a suicide gene.
  • a gene such as the herpes simplex thymidine kinase (TK) gene can be transferred into tumor cells to induce their destruction by administration of acyclovir or gancyclovir.
  • the TK expressing tumor cells can present their tumor antigens, and are capable of proliferation.
  • the cells can be selectively killed. This approach might allow longer viability of the tumor cells utilized for immunizations, which may be advantageous in the induction or augmentation of anti-tumor immunity.
  • CE cells and/or carrier cells and tumor cells, and/or homogenates of tumor cells and/or purified tumor antigen(s), are combined for patient immunization. Approximately 10 7 tumor cells will be required. If homogenates of tumor cells or purified or non-purified fractions of tumor antigens are used, the tumor dose can be adjusted based on the normal number of tumor antigens usually present on 10 7 intact tumor cells.
  • the tumor preparation should be mixed with numbers of CE or carrier cells sufficient to secrete cytokine levels that induce anti-tumor immunity (11-12) without producing substantial systemic toxicity which would interfere with therapy.
  • the cytokines should be produced by the CE cells or the carrier cells at levels sufficient to induce or augment immune response but low enough to avoid substantial systemic toxicity. This prevents side effects created by previous methods' administration of greater than physiological levels of the cytokines.
  • formulations will be formulated for injection in any manner known in the art acceptable for immunization. Because it is important that at least the CE cells and carrier cells remain viable, the formulations must be compatible with cell survival. Formulations can be injected subcutaneously, intramuscularly, or in any manner acceptable for immunization.
  • Contaminants in the preparation which may focus the immune response on undesired antigens should be removed prior to the immunizations.
  • Skin punch biopsies will be obtained from each patient under sterile conditions.
  • the biopsy tissue will be minced and placed in RPMI 1640 media containing 10% fetal calf serum (or similar media) to establish growth of the skin fibroblasts in culture.
  • the cultured fibroblasts will be utilized to generate IL-2 secreting CE cells by retroviral mediated IL-2 gene transfer.
  • the cultured skin fibroblasts will then be infected with a retroviral vector containing the IL-2 and Neomycin resistance (Neo R ) genes.
  • An N2 vector containing the Neo R gene will be used, and has been previously utilized by a number of investigators for in vitro and in vivo work, including investigations with human subjects (16).
  • the IL-2 vector will be generated from an N2-derived vector, LLRNL, developed and described by Friedmann and his colleagues (20). It will be made by replacement of the luciferase gene of LLRNL with a full-length cDNA encoding human IL-2.
  • Retroviral vector free of contaminating replication-competent virus is produced by transfection of vector plasmid constructions into the helper-free packaging cell line PA317.
  • the vector Before infection of patients' cells, the vector will have been shown to be free of helper virus. In the event that helper virus is detected, the vector will be produced in the GP+envAM12 packaging cell line in which the viral gag and pol genes are separated from the env, further reducing the likelihood of helper virus production.
  • the cultured primary fibroblasts will be incubated with supernatant from the packaging cell line as described (20). Supernatant from these cells will be tested for adventitious agents and replication competent virus as described (16) and outlined in Table 1.
  • the fibroblasts are washed and then grown in culture media containing G418, (a neomycin analogue) to select for transduced cells expressing the Neo R gene.
  • the G418-resistant cells will be tested for expression of the IL-2 gene by measuring the concentration of IL-2 in the culture supernatant by an enzyme linked immunosorbent assay (ELISA) (12). G418-resilient cells expressing IL-2 will be stored at -70° C. until required for subsequent use in immunizations.
  • ELISA enzyme linked immunosorbent assay
  • Tumors obtained form clinically indicated surgical resections or from superficial lymph node or skin metastases will be minced into 2-3 mm pieces and treated with collagenase and DNAse to facilitate separation of the tumor into a single cell suspension.
  • the collected cells will be centrifuged and washed in RPMI 1640 media and then cryopreserved in a solution containing 10% dimethyl sulphoxide and 50% fetal calf serum in RPMI 1640 media.
  • the cells will be stored in liquid nitrogen until the time of administration.
  • the cells Prior to their use in subcutaneous immunizations, the cells will be thawed, washed in media free of immunogenic contaminants, and irradiated with 4,000 rads per minute for a total of 20,000 rads in a cesium irradiator.
  • Patients will have a histologically confirmed diagnosis of cancer. Patients with tumors that must be resected for therapeutic purposes or with tumors readily accessible for biopsy are most appropriate for this embodiment of the invention.
  • CBC with differential, platelet count, PT, PTT, glucose, BUN, creatinine, electrolytes, SGOT, SGPT, LDH, alkaline phosphatase, bilirubin, uric acid, calcium, total protein albumin.
  • Chest X-ray and other diagnostic studies including computerized tomography (CT), magnetic resonance imaging (MRI) or radionuclide scans may be performed to document and quantify the extent of disease activity.
  • CT computerized tomography
  • MRI magnetic resonance imaging
  • radionuclide scans may be performed to document and quantify the extent of disease activity.
  • Each patient will receive subcutaneous immunizations with a mixture if irradiated tumor cells and autologous fibroblast CE cells genetically modified to secrete IL-2.
  • Approximately 10 7 tumor cells will be mixed with 10 7 fibroblasts known to secrete at least 20 units/ml of IL-2 in tissue culture when semi-confluent (12).
  • the irradiated tumor cells and genetically modified fibroblasts will be placed in a final volume of 0.2 ml normal saline for immunization.
  • At least two subcutaneous immunizations will be administered, two weeks apart, with irradiated tumor cells and autologous fibroblasts genetically modified to secrete IL-2. If no toxicity is observed, subsequent booster immunizations may be administered periodically (at least one week apart) to optimize the anti-tumor immune response.
  • any resulting uric acid nephropathy, adult respiratory distress syndrome, disseminated intravascular coagulation or hyperkalemia will be treated using standard methods.
  • Hypersensitivity reactions such as chills, fever and/or rash will be treated symptomatically with antipyretics and antihistamines. Patients should not be treated prophylactically. Should arthralgias, lymphadenopathy or renal dysfunction occur, treatment with corticosteroids and/or antihistamines will be instituted. Anaphylaxis will be treated by standard means such as administration of epinephrine, fluids, and steroids.
  • Retroviral vectors were employed to transfer and express IL-2 and neomycin phosphotransferase genes in murine and primary human fibroblasts.
  • the retroviral vector DC/TKIL2 produced by Gilboa and co-workers (Gansbacher, et al., J. Exp. Med. 172:1217-1223, 1990, which is incorporated herein by reference) was utilized to transduce murine fibroblasts for application in an animal tumor model (see Section B below).
  • Human fibroblasts were transduced with the retroviral vector LXSN-RI-IL2 (SEQ ID NO:1). Schematic diagrams of the structure of these retroviral vectors are provided in FIG. 1.
  • a more complete description of the LXSN-RI-IL2 vector, including its nucleotide sequence, is provided in Example III and in Tables 2, 3, 4, 5 and 6 (SEQ ID NO:1).
  • FIG. 2 depicts the levels of IL-2 secreted by the transduced fibroblasts.
  • Biological activity of the IL-2 expressed by the transduced human fibroblasts was confirmed by a cell proliferation bioassay employing an IL-2 dependent T cell line.
  • a cell proliferation bioassay employing an IL-2 dependent T cell line.
  • supernatant from the transduced fibroblasts and control unmodified fibroblasts were incubated with the IL-2 dependent T cell line CTLL-2.
  • Incorporation of 3 H-thymidine was measured as an indicator of cell proliferation and IL-2 activity (FIG. 3).
  • fibroblasts genetically modified to secrete IL-2 was tested in an animal model of colorectal carcinoma.
  • the Balb/c CT26 tumor cell line was injected subcutaneously with Balb/c fibroblasts transduced to express IL-2.
  • Control groups included animals injected with 1) a mixture of CT26 tumor cells and unmodified fibroblasts; 2) CT26 tumor cells without fibroblasts and 3) transduced fibroblasts alone. No tumors were detected in 3/8 animals treated with transduced fibroblasts and CT26 cells. In contrast, all untreated control animals (8/8) injected with CT26 tumor cells developed palpable tumors. No tumors were detected in the animals inoculated with transduced fibroblasts without CT26 tumor cells.
  • the mean CT26 tumor size in Balb/c mice injected with the IL-2 secreting fibroblasts was considerably smaller compared to the control groups (FIG. 4).
  • a multivariate non-parametric statistical procedure (Koziol, et al., Biometries 37:383-390, 1981 and Koziol, et al., Computer Prog. Biomed. 19:69-74, 1984, which is incorporated herein by reference) was utilized to evaluate differences in tumor growth among the treatment groups.
  • Lymphokine gene therapy of cancer will be evaluated in cancer patients who have failed conventional therapy.
  • An N2-derived vector containing the neomycin phosphotransferase gene will be used. This vector has been employed by a number of investigators for in vitro and in vivo studies including recently approved investigations with human subjects (Rosenberg et al., N. Eng. J. Med., 323:570-578, 1990).
  • the lymphokine vectors used in this investigation will be generated from the N2-derived vector, LXSN, developed and described by Miller et al., Mol. Cell Biol. 6:2895, 1986 and Miller et al., BioTechniques 7:980, 1989, which are incorporated herein by reference.
  • the vector LXSN-RI-IL2 contains human IL-2 cDNA under the control of the retroviral 5' LTR promoter and the neomycin phosphotransferase gene under the control of the SV40 promoter (see FIG. 1).
  • the normal human IL-2 leader sequence has been replaced with a chimeric sequence containing rat insulin and human IL-2 leader sequences (see Tables 2, 3, 4, 5 and 6 (SEQ ID NO:1)). This chimeric leader sequence enhances IL-2 gene expression.
  • the bacterial plasmid pBC12/CMV/IL2 (Cullen, B. R., DNA 7:645-650, 1988, which is incorporated herein by reference) containing the full-length IL-2 cDNA and chimeric leader sequence was digested with HindIII and the ends were blunted using Klenow polymerase.
  • IL-2 cDNA was subsequently released from the plasmid by digestion with BamHI.
  • the IL-2 fragment was purified by electrophoresis in a 1% agarose gel and the appropriate band was extracted utilizing a glass powder method. Briefly, the gel slice was dissolved in 4M NaI at 55°.
  • pLXSN-RI-IL2 DNA was transfected into the ecotropic packaging cell line PE501 by standard calcium phosphate precipitation methods (Miller et al., Mol. Cell Biol. 6:2895, 1986).
  • the transfected PE501 cell line was grown in DMEM medium with 10% FCS. The medium was changed after 24 hours and supernatant harvested 24 hours later to infect the amphotropic packaging cell line PA317 as described (Miller et al., Mol. Cell Biol. 6:2895, 1986 and Miller et al., BioTechniques 7:980, 1989).
  • the infected PA317 cells were harvested by trypsinization 24 hours later and replated 1:20 in DMEM containing 10% FCS and the neomycin analogue G418 (400 ⁇ g/ml). The cells were grown at 37° C. in 7% CO 2 atmosphere. The selection medium was changed every 5 days until colonies appeared. On day 14, twenty colonies were selected, expanded and tested for vital production by standard methods (Xu et al., Virology 171:331-341, 1989). Briefly, supernatants were harvested from confluent culture dishes, passed through a 0.45 ⁇ m filter, diluted with DMEM with 10% FCS and utilized to infect NIH 3T3 cells in the presence of 8 ⁇ g/ml polybrene.
  • the infected NIH 3T3 cells were grown in culture medium that contained the neomycin analogue G418. After 12-14 days, the colonies were stained, counted and the viral titer calculated as described (Xu et al., Virology 171:331-341, 1989).
  • Colonies with the highest viral titers (>10 4 infectious units/ml) were tested for IL-2 expression by Northern blot analyses. Colonies with the highest viral titers and documented IL-2 expression were cryopreserved and will be utilized as stock cultures to produce the LXSN-RI-IL2 retroviral vector trial.
  • LXSN-IL2 and LNCX-IL2 were packaged in the PA317 cell line for production of retroviral supernatant.
  • the high level expressing, double copy vector DC/TKIL-2 vector thymidine kinase promoter
  • the culture conditions for the growth of primary fibroblasts retroviral transduction were optimized. Primary fibroblasts were successfully cultured. The optimal conditions enable the growth of approximately 3-4 ⁇ 10 6 primary fibroblasts from a 12 mm 2 skin biopsy in approximately 4-6 weeks. Retroviral infection, G418 selection, and expansion of the genetically modified fibroblasts takes an additional 4-6 weeks.
  • fibroblasts are synchronized in G1 phase by serum starvation, followed by stimulation with medium containing 15% fetal bovine serum 15 hours prior to transduction.
  • the cells are then subjected to 2 cycles of retrovirus infection, each cycle lasting approximately 3 hours.
  • the cells are refed with fresh media overnight, and then selection in G418 is initiated the next day.
  • This method is capable of transducing 5-15% of the fibroblasts in a culture, depending on the multiplicity of infection.
  • Table 8 compares the expression levels of IL-2 in fibroblast lines transduced with LXSN-IL2.
  • IL-2 expression levels in established, embryonic, and primary fibroblast cultures are similar. Comparison of these data with Table 7 suggest that IL-2 expression is affected more by factors such as different promoters than by the fibroblast line used. Similarly, changes in culture conditions can have important effects on IL-2 expression. Table 9 shows that transduced GT1 cells, a primary human fibroblast culture expressed 15-fold more IL-2 under 100 ⁇ g/ml G418 selection than under 25 ⁇ g/ml G418 selection. Several other primary fibroblast lines have also been transduced with our vectors and are currently growing under G418 selection.
  • nPBL peripheral blood lymphocytes
  • the results of each experiment are presented with two figures and one table.
  • the rate of tumor growth for each treatment group is presented as the mean tumor size in the group over time.
  • a Kaplan-Meier curve presents the time of tumor onset for the individual animals in each treatment group.
  • the number of animals, the number and percentage of tumor free animals, and the tumor size distribution patterns for each experiment are presented in a table.
  • mice were injected subcutaneously with mixtures of 5 ⁇ 10 4 CT26 cells and 2 ⁇ 10 6 fibroblasts genetically modified by different retroviral vectors to express IL-2.
  • 31 of 33 animals 9
  • 22 out of the 34 animals (65%) receiving fibroblast mediated cytokine gene therapy were tumor free at 3 weeks, and 5 animals (18%) remain tumor free after 12 weeks.
  • Those animals that received fibroblast mediated IL-2 therapy and developed tumor were characterized by a delayed onset and rate of tumor growth.
  • LNCX-IL2 the highest expressing vector caused substantially greater inhibition of tumorigenicity than the lower expressing vector DC/TKIL-2.
  • Subsequent comparisons between the control arm and animals that received tumor cells mixed with IL-2 transduced fibroblasts revealed a significant difference (P ⁇ 0.05).
  • mice When mice were injected with 2 ⁇ 10 6 modified fibroblasts mixed with 1 ⁇ 10 5 live tumor cells the results became more striking (see FIGS. 8 and 9, and Table 12). All the control animals developed tumors after 4 weeks whereas 33% and 27% of the animals treated with fibroblasts modified with the DCTK-IL2 or LXSN-IL2 vectors (respectively) remain tumor free after 7 weeks (the experiment is ongoing). More dramatically, 75% of the animals treated with fibroblasts modified with the highest IL-2 producing vector, LNCX-IL2, remain tumor free after 7 weeks. These data clearly demonstrate the importance of an initial high dose of IL-2 to prevent tumor establishment.
  • mice were injected with CT26 cells genetically modified to express IL-2 (results not shown). Injection of up to 1 ⁇ 10 6 IL-2 expressing tumor cells into Balb/c mice failed to produce tumors. Injection of higher numbers however, resulted in some animals developing tumors with delayed onset. These data confirm the results reported in the literature (1).
  • CT26 tumor cells modified with the DCTK-IL2 vector with 1 ⁇ 10 5 unmodified tumor cells.
  • FIGS. 10 and 11, and Table 13 show that DCTK-IL2 modified tumor cells are somewhat effective in preventing tumor development.
  • the mean tumor size for the treatment arm is 303 mm 2 , compared to 620 mm 2 for the control arm. After 22 weeks, one animal (10%) remains tumor free, compared to none in the control arms. Data for animals treated under the same conditions with DCTK-IL2 modified fibroblasts in a separate experiment are included for comparison purposes. This comparison suggests that DCTK-IL2 modified tumor cells have an effect on tumor establishment similar to that of DCTK-IL2 modified fibroblasts.
  • mice were immunized with 2.5 ⁇ 10 5 irradiated tumor cells either alone or mixed with 2 ⁇ 10 6 transduced or unmodified fibroblasts, and challenged one week later with 5 ⁇ 10 4 live tumor cells in the opposite flank.
  • results demonstrate that immunization with irradiated tumor cells and transduced fibroblasts protect some animals against a live tumor challenge, but that the protection is only slightly better than that achieved by immunization with irradiated tumor cells alone or irradiated tumor cells mixed with unmodified fibroblasts.
  • the mean tumor size of this group was 41 mm 2 , compared to 180, 170, and 140 mm 2 for the three control groups.
  • Animals treated with LNCX-IL2 modified fibroblasts were also protected against subsequent tumor challenge, but the results were less striking.
  • 54% of the animals remain tumor free and the mean tumor size for the group at four weeks was 86 mm 2 .
  • the number of tumor free animals in the group treated with LXSN-IL2 modified fibroblasts was similar to the control groups, although the tumors were slightly delayed in their onset.
  • a multivariate non-parametric statistical procedure (19, 20), utilized to evaluate differences in tumor onset, demonstrated that the differences for the six arms presented in FIG. 15 were significant (p 0.012).

Abstract

A novel method of tumor immunotherapy is described comprising the genetic modification of cells resulting in the secretion of cytokine gene products to stimulate a patient's immune response to tumor antigens. In one embodiment, autologous fibroblasts genetically modified to secrete at least one cytokine gene product are utilized to immunize the patient in a formulation with tumor antigens at a site other than an active tumor site. In another embodiment, cells genetically modified to express at least one tumor antigen gene product and to secrete at least one cytokine gene product are utilized in a formulation to immunize the patient at a site other than an active tumor site.

Description

This application is a continuation of application Ser. No. 07/863,641, filed Apr. 3, 1992, now abandoned, which is a continuation-in-part of U.S. patent application Ser. No. 07/781,356, filed on Oct. 25, 1991, now abandoned, which is a continuation-in-part of U.S. patent application Ser. No. 07/720,872, filed on Jun. 25, 1991, now abandoned, both of which are incorporated herein in their entirety.
BACKGROUND
Recent advances in our understanding of the biology of the immune system have lead to the identification of important modulators of immune responses, called cytokines (1-3). Immune system modulators produced by lymphocytes are termed lymphokines, a subset of the cytokines. These agents mediate many of the immune responses involved in anti-tumor immunity. Several of these cytokines have been produced by recombinant DNA methodology and evaluated for their anti-tumor effects. The administration of lymphokines and related immunomodulators has resulted in objective tumor responses in patients with various types of neoplasms (4-7). However, current modes of cytokine administration are frequently associated with toxicities that limit the therapeutic value of these agents.
For example, interleukin-2 (IL-2) is an important lymphokine in the generation of anti-tumor immunity (4). In response to tumor antigens, a subset of lymphocytes termed helper T-cells secrete small quantities of IL-2. This IL-2 acts locally at the site of tumor antigen stimulation to activate cytotoxic T-cells and natural killer cells which mediate systemic tumor cell destruction. Intravenous, intralymphatic and intralesional administration of IL-2 has resulted in clinically significant responses in some cancer patients (4-6). However, severe toxicities (hypotension and adema) limit the dose and efficacy of intravenous and intralymphatic IL-2 administration (5-7). The toxicity of systemically administered lymphokines is not surprising as these agents mediate local cellular interactions and they are normally secreted in only very small quantities.
Additionally, other cytokines, such as interleukin-4 (IL-4), alpha interferon (α-INF) and gamma interferon (c-INF) have been used to stimulate immune responses to tumor cells. Like IL-2, the current modes of administration have adverse side effects.
To circumvent the toxicity of systemic cytokine administration, several investigators have examined intralesional injection of IL-2. This approach eliminates the toxicity associated with systemic IL-2 administration (8,9,10). However, multiple intralesional injections are required to optimize therapeutic efficacy (9,10). Hence, these injections are impractical for many patients, particularly when tumor sites are not accessible for injection without potential morbidity.
An alternative approach, involving cytokine gene transfer into tumor cells, has resulted in significant anti-tumor immune responses in several animal tumor models (11-14). In these studies, the expression of cytokine gene products following cytokine gene transfer into tumor cells has abrogated the tumorigenicity of the cytokine-secreting tumor cells when implanted into syngeneic hosts. The transfer of genes for IL-2 (11,12) c-INF (13) or interleukin-4 (IL-4) (14) significantly reduced or eliminated the growth of several different histological types of murine tumors. In the studies employing IL-2 gene transfer, the treated animals also developed systemic anti-tumor immunity and were protected against subsequent tumor challenges with the unmodified parental tumor (11,12). Similar inhibition of tumor growth and protective immunity was also demonstrated when immunizations were performed with a mixture of unmodified parental tumor cells and genetically modified tumor cells engineered to express the IL-2 gene. No toxicity associates with localized lymphokine transgene expression was reported in these animal tumor studies (11-14).
While the above gene-transfer procedure has been shown to provide anti-tumor immunity, it still retains practical difficulties. This approach is limited by the inability to transfer functional cytokine genes into many patients' tumor cells, as most patients' tumors cannot be established to grown in vitro and methods for human in vivo gene transfer are not available.
SUMMARY OF THE INVENTION
The present invention demonstrates a novel, more practical method of cytokine cancer immunotherapy. In one approach, selected cells from a patient, such as fibroblasts, obtained, for example, from a routine skin biopsy, are genetically modified to express one or more cytokines. Alternatively, patient cells which may normally serve as antigen presenting cells in the immune system such as macrophages, monocytes, and lymphocytes may also be genetically modified to express one or more cytokines. These modified cells are hereafter called cytokine-expressing cells, ore CE cells. The CE cells are then mixed with the patient's tumor antigens, for example in the form of irradiated tumor cells, or alternatively in the form of purified natural or recombinant tumor antigen, and employed in immunizations, for example subcutaneously, to induce systemic anti-tumor immunity.
The cytokines are locally expressed at levels sufficient to induce or augment systemic anti-tumor immune responses via local immunization at sites other than active tumor sites. Systemic toxicity related to cytokine administration should not occur because the levels of cytokine secreted by the CE cells should not significantly affect systemic cytokine concentrations.
As the amount of cytokine secreted by the CE cells is sufficient to induce anti-tumor immunity but is too low to produce substantial systemic toxicity, this approach provides the benefit of local cytokine administration. In addition, this novel method obviates the need for intralesional injections, which may produce morbidity. Furthermore, the continuous local expression of cytokine(s) at the sites of immunization may also augment anti-tumor immune responses compared to intermittent cytokine injections. This method also provides the advantage of local immunization with the CE cells, as opposed to cumbersome intravenous infusions. This method also eliminates the need for establishing tumor cell lines in vitro as well as transfer of genes into these tumor cells.
This invention also provides an alternative means of localized expression of cytokines to induce and/or increase immune responses to a patient's tumor through genetic modification of cellular expression of both cytokine(s) and tumor antigen(s). In this embodiment, selected cells from a patient are isolated and transduced with cytokine gene(s) as well as gene(s) coding for tumor antigen(s). The transduced cells are called "carrier cells." Carrier cells can include fibroblasts and cells which may normally serve as antigen presenting cells in the immune system such as macrophages, monocytes, and lymphocytes. Transduced carrier cells actively expressing both the cytokine(s) and the tumor antigen(s) are selected and utilized in local immunizations at a site other than active tumor sites to induce anti-tumor immune responses. As with the CE cells, these carrier cells should not produce substantial systemic toxicities, as the levels of cytokine(s) secreted by the carrier cells should not significantly affect systemic cytokine concentrations. This alternate embodiment is advantageous because it obviates the need to obtain samples of the tumor, which is sometimes difficult. However, carrier cells can be utilized in local immunizations in conjunction with tumor cells, tumor cell homogenates, purified tumor antigens, or recombinant tumor antigens to enhance anti-tumor immunity.
Additionally, this second embodiment retains the same advantages as the first embodiment in that the level of cytokine released by the carrier cells is sufficient to induce anti-tumor immunity but is too low to produce substantial systemic toxicity. In addition, as with the first embodiment, this method obviates the need for intralesional injections, and allows for continuous expression of cytokine(s). This method also eliminates the need for establishing continuous cultures in vitro of tumor cells as well as transfer of genes into these tumor cells, and provides the advantage of local immunization with the carrier cells, as opposed to cumbersome lengthy intravenous infusions.
These approaches may also find application in inducing or augmenting immune responses to other antigens of clinical significance in other areas of medical practice.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows schematic diagrams of retroviral vectors DC/TKIL2, LXSN-IL2, and LNCX-IL2.
FIG. 2 shows a mean IL-2 concentration of triplicate supernatant samples measured by ELISA. Supernatants were harvested from overnight cultures of approximately 1.5×106 semi-confluent fibroblasts.
FIG. 3 shows biological activity of the IL-2 secreted by the transduced fibroblasts was demonstrated by measuring mean 3 H-TdR incorporation of an IL-2 dependent T-cell line incubated with triplicate samples of supernatants. Supernatants were harvested from overnight cultures of approximately 1.5×106 semi-confluent fibroblasts.
FIG. 4 shows comparisons between animals injected with 105 CT26 tumor cells alone (□); 105 CT26 tumor cells and 2×106 unmodified BALB/C fibroblasts (▪); 105 CT26 tumor cells and 2×106 IL-2 transduced BALB/C fibroblasts (); and 105 CT26 tumor cells and 1×106 transduced BALB/C fibroblasts (◯). Tumor measurements are the mean products of the cross-sectional diameter of the tumors from four animals in each treatment group. The (*) indicates statistically significant difference (P<0.05) in tumor growth curves.
FIG. 5 shows PCR analysis of neomycin phosphotransferase DNA sequences. Lane 1--positive control pLXSN-RI-IL2. Lanes 2 through 4 tests genomic DNA; Lanes 5 and 6 ovary genomic DNA; Lane 7 negative control, no DNA. Identical results were obtained with liver, spleen and lung genomic DNA (data not shown).
FIG. 6 shows the effect of IL-2 modified fibroblasts on tumor establishment and development using 2×106 fibroblasts mixed with 5×104 CT26 tumor cells concentrating on the rate of tumor growth.
FIG. 7 shows the effect of IL-2 modified fibroblasts on tumor establishment and development using 2×106 fibroblasts mixed with 5×104 CT26 tumor cells concentrating on the time of tumor onset for the individual animal in each treatment group.
FIG. 8 shows the effect of IL-2 modified fibroblasts on tumor establishment and development using 2×106 fibroblasts mixed with 1×105 CT26 tumor cells concentrating on the rate of tumor growth.
FIG. 9 shows the effect of IL-2 modified fibroblasts on tumor establishment and development using 2×106 fibroblasts mixed with 1×105 CT26 tumor cells concentrating on the time of tumor onset for the individual animal in each treatment group.
FIG. 10 shows the effect of IL-2 modified cells on tumor establishment and development using 2×106 DCTK-IL2-modified CT26 tumor cells mixed with 1×105 unmodified CT26 compared to 2×106 DCTK-IL2-modified fibroblasts mixed with 1×105 CT26 concentrating on the rate of tumor growth.
FIG. 11 shows the effect of IL-2 modified cells on tumor establishment and development using 2×106 DCTK-IL2-modified CT26 tumor cells mixed with 1×105 unmodified CT26 compared to 2×106 DCTK-IL2-modified fibroblasts mixed with 1×105 CT26 concentrating on the time of tumor onset for the individual animal in each treatment group.
FIG. 12 shows the effect of IL-2 modified fibroblasts on induction of systemic anti-tumor immunity and the rate of tumor growth. Mice were immunized with 2×106 fibroblasts mixed with 2.5×105 irradiated CT26 tumor cells 7 days prior to challenge with 5×104 fresh tumor cells.
FIG. 13 shows the effect of IL-2 modified fibroblasts on induction of systemic anti-tumor immunity and the time of tumor onset for the individual animal in each treatment group. Mice were immunized with 2×106 fibroblasts mixed with 2.5×105 irradiated CT26 tumor cells 7 days prior to challenge with 5×104 fresh tumor cells.
FIG. 14 shows the effect of IL-2 modified fibroblasts on induction of systemic anti-tumor immunity and the rate of tumor growth. Mice were immunized with 2×106 fibroblasts mixed with 2.5×105 irradiated CT26 tumor cells 14 days prior to challenge with 5×104 fresh tumor cells.
FIG. 15 shows the effect of IL-2 modified fibroblasts on induction of systemic anti-tumor immunity and the time of tumor onset for the individual animal in each treatment group. Mice were immunized with 2×106 fibroblasts mixed with 2.5×105 irradiated CT26 tumor cells 14 days prior to challenge with 5×104 fresh tumor cells.
DETAILED DESCRIPTION
A novel method of tumor immunotherapy is described comprising the genetic modification of cells resulting in the secretion of cytokine gene products to stimulate a patient's immune response to tumor antigens. "Gene" is defined herein to be a nucleotide sequence encoding the desired protein. In one embodiment, autologous fibroblasts genetically modified to secrete at least one cytokine gene product are utilized to immunize the patient in a formulation with tumor antigens at a site other than an active tumor site. In another embodiment, cells genetically modified to express at least one tumor antigen gene product and to secrete at least one cytokine gene product are utilized in formulation to immunize the patient at a site other than an active tumor site. Cytokines are preferably expressed in cells which efficiently secrete these proteins into the surrounding milieu. Fibroblasts are an example of such cells. Fibroblasts or other cells can be genetically modified to express and secrete one or more cytokines, as described later in this specification.
Tumor antigens can be provided by several methods, including, but not limited to the following: 1) CE cells can be transduced with gene(s) coding for tumor antigens. These "carrier cells" are then utilized in patient immunizations. 2) Cloned gene sequences coding for appropriate tumor antigens can be transferred into cells such as fibroblasts or antigen-presenting cells. These cells are then mixed with CE or carrier cells to immunize the patient. 3) Tumor antigens can be cloned in bacteria or other types of cells by recombinant procudures. These antigens are then purified and employed an immunization with CE and/or carrier cells. 4) Tumor antigens can be purified from tumor cells and used, along with CE or carrier cells, to immunize the patient. 5) Tumor cells may be irradiated or mechanically disrupted and mixed with CE and/or carrier cells for patient immunizations.
This invention encompasses the following steps: (A) isolation of appropriate cells for generation of CE cells or carrier cells; (B) isolation of cytokine genes or isolation of cytokine genes and tumor antigen genes, as well as appropriate marker and/or suicide genes; (C) transfer of the genes from (B) to produce the CE cells or carrier cells; (D) preparation of immunological samples of the patient's tumor antigens or other suitable tumor antigens for immunization with CE or carrier cells; (E) inactivation of the malignant potential of tumor cells if they are used as a source of tumor antigens for immunization; and (F) preparation of samples for immunization. Following are several embodiments contemplated by the inventors. However, it is understood that any means known by those in the art to accomplish these steps will be usable in this invention.
(A) Isolation of Cells to Generate CE and Carrier Cells
Cells to be utilized as CE cells and carrier cells can be selected from a variety of locations in the patient's body. For example, skin punch biopsies provide a readily available source of fibroblasts for use in generating CE cells, with a minimal amount of intrusion to the patient. Alternatively, these fibroblasts can be obtained from the tumor sample itself. Cells of hematopoietic origin may be obtained by venipuncture, bone marrow aspiration, lymph node biopsies, or from tumor samples. Other appropriate cells for the generation of CE or carrier cells can be isolated by means known in the art. Non-autologous cells similarly selected and processed can also be used.
(B) Isolation of Genes
Numerous cytokine genes have been cloned and are available for use in this protocol. The genes for IL-2, c-INF and other cytokines are readily available (1-5, 11-14). Cloned genes of the appropriate tumor antigens are isolated according to means known in the art.
Selectable marker genes such as neomycin resistance (NeoR) are readily available. Incorporation of a selectable marker gene(s) allows for the selection of cells that have successfully received and express the desired genes. Other selectable markers known to those in the art of gene transfer may also be utilized to generate CE cells or carrier cells expressing the desired transgenes.
"Suicide" genes can be incorporated into the CE cells or carrier cells to allow for selective inducible killing after stimulation of the immune response. A gene such as the herpes simplex virus thymidine kinase gene (TK) can be used to create an inducible destruction of the CE cells or carrier cells. When the CE cells or carrier cells are no longer useful, a drug such as acyclovir or gancyclovir can be administered. Either of these drugs will selectively kill cells expressing TK, thus eliminating the implanted transduced cells. Additionally, a suicide gene may be a gene coding for a non-secreted cytotoxic polypeptide attached to an inducible promoter. When destruction of the CE or carrier cells is desired, the appropriate inducer of the promoter is administered so that the suicide gene is induced to produce cytotoxic polypeptide which subsequently kills the CE or carrier cell. However, destruction of the CE or carrier cells may not be required.
Genes coding for tumor antigen(s) of interest can be cloned by recombinant methods. The coding sequence of an antigen expressed by multiple tumors may be utilized for many individual patients.
(C) Transfer of Genes
Numerous methods are available for transferring genes into cultured cells (15). For example, the appropriate genes can be inserted into vectors such as plasmids or retroviruses and transferred into the cells. Electroporation, lipofection and a variety of other methods are known in the field and can be implemented.
One method for gene transfer is a method similar to that employed in previous human gene transfer studies, where tumor infiltrating lymphocytes (TILs) were modified by retroviral gene transduction and administered to cancer patients (16). In this Phase I safety study of retroviral mediated gene transfer, TILs were genetically modified to express the Neomycin resistance (NeoR) gene. Following intravenous infusion, polymerase chain reaction analyses consistently found genetically modified cells in the circulation for as long as two months after administration. No infectious retroviruses were identified in these patients and no side effects due to gene transfer were noted in any patients (16). These retroviral vectors have been altered to prevent viral replication by the deletion of viral gag, pol and env genes.
When retroviruses are used for gene transfer, replication competent retroviruses may theoretically develop by recombination between the retroviral vector and viral gene sequences in the packaging cell line utilized to produce the retroviral vector. We will use packaging cell lines in which the production of replication competent virus by recombination has been reduced or eliminated. Hence, all retroviral vector supernatants used to infect patient cells will be screened for replication competent virus by standard assays such as PCR and reverse transcriptase assays (16). Furthermore, exposure to replication competent virus may not be harmful. In studies of subhuman primates injected with a large inoculum of replication competent murine retrovirus, the retrovirus was cleared by the primate immune system (17). No clinical illnesses or sequelae resulting from replication competent virus have been observed three years after exposure. In summary, it is not expected that patients will be exposed to replication competent murine retrovirus and it appears that such exposure may not be deleterious (17).
(D) Preparation of Immunological Samples of the Patient's Tumor Antigens or Purified Recombinant Tumor Antigens
Tumor cells bearing tumor associated antigens are isolated from the patient. These cells can derive either from solid tumors or from leukemic tumors. For solid tumors, single-cell suspensions can be made by mechanical separation and washing of biopsy tissue (18).
Hematopoietic tumors may be isolated from peripheral blood or bone marrow by standard methods (19).
A second variant is the use of homogenates of tumor cells. Such homogenates would contain tumor antigens available for recognition by the patient's immune system upon stimulation by this invention. Either unfractionated cell homogenates, made, for example, by mechanical disruption or by freezing and thawing the cells, or fractions of homogenates preferably with concentrated levels of tumor antigens, can be used.
Likewise, purified tumor antigens, obtained for example by immunoprecipitation or recombinant DNA methods, could be used. Purified antigens would then be utilized for immunizations together with the CE cells and/or carrier cells described above to induce or enhance the patient's immune response to these antigens.
In the embodiments employing carrier cells, tumor antigens are available through their expression by the carrier cells. These carrier cells can be injected alone or in conjunction with other tumor antigen preparations or CE cells. Likewise, when CE cells are used, purified recombinant tumor antigen, produced by methods known in the art, can be used.
If autologous tumor cells are not readily available, heterologous tumor cells, their homogenates, their purified antigens, or carrier cells expressing such antigens could be used.
(E) Inactivation of Tumor Cells
When viable tumor cells are utilized in immunizations as a source of tumor antigens, the tumor cells can be inactivated so that they do not grow in the patient. Inactivation can be accomplished by several methods. The cells can be irradiated prior to immunization (18). This irradiation will be at a level which will prevent their replication. Such viable calls can then present their tumor antigens to the patient's immune system, but cannot multiply to create new tumors.
Alternatively, tumor cells that can be cultured may be transduced with a suicide gene. As described above, a gene such as the herpes simplex thymidine kinase (TK) gene can be transferred into tumor cells to induce their destruction by administration of acyclovir or gancyclovir. After immunization, the TK expressing tumor cells can present their tumor antigens, and are capable of proliferation. After a period of time during which the patients's immune response is stimulated, the cells can be selectively killed. This approach might allow longer viability of the tumor cells utilized for immunizations, which may be advantageous in the induction or augmentation of anti-tumor immunity.
(F) Preparation of Samples for Immunization
CE cells and/or carrier cells and tumor cells, and/or homogenates of tumor cells and/or purified tumor antigen(s), are combined for patient immunization. Approximately 107 tumor cells will be required. If homogenates of tumor cells or purified or non-purified fractions of tumor antigens are used, the tumor dose can be adjusted based on the normal number of tumor antigens usually present on 107 intact tumor cells. The tumor preparation should be mixed with numbers of CE or carrier cells sufficient to secrete cytokine levels that induce anti-tumor immunity (11-12) without producing substantial systemic toxicity which would interfere with therapy.
The cytokines should be produced by the CE cells or the carrier cells at levels sufficient to induce or augment immune response but low enough to avoid substantial systemic toxicity. This prevents side effects created by previous methods' administration of greater than physiological levels of the cytokines.
These mixtures, as well as carrier cells that are utilized alone, will be formulated for injection in any manner known in the art acceptable for immunization. Because it is important that at least the CE cells and carrier cells remain viable, the formulations must be compatible with cell survival. Formulations can be injected subcutaneously, intramuscularly, or in any manner acceptable for immunization.
Contaminants in the preparation which may focus the immune response on undesired antigens should be removed prior to the immunizations.
The following examples are provided for illustration of several embodiments of the invention and should not be interpreted as limiting the scope of the invention.
EXAMPLE I Immunization with Fibroblasts Expressing IL-2 Mixed with Irradiated Tumor Cells
1) Isolation of Autologous Fibroblasts for Use in Generating IL-2 Secreting CE Cells
Skin punch biopsies will be obtained from each patient under sterile conditions. The biopsy tissue will be minced and placed in RPMI 1640 media containing 10% fetal calf serum (or similar media) to establish growth of the skin fibroblasts in culture. The cultured fibroblasts will be utilized to generate IL-2 secreting CE cells by retroviral mediated IL-2 gene transfer.
2) Retrovital Vector Preparation and Generation of IL-2 Secreting CE Cells
The cultured skin fibroblasts will then be infected with a retroviral vector containing the IL-2 and Neomycin resistance (NeoR) genes. An N2 vector containing the NeoR gene will be used, and has been previously utilized by a number of investigators for in vitro and in vivo work, including investigations with human subjects (16). The IL-2 vector will be generated from an N2-derived vector, LLRNL, developed and described by Friedmann and his colleagues (20). It will be made by replacement of the luciferase gene of LLRNL with a full-length cDNA encoding human IL-2. Retroviral vector free of contaminating replication-competent virus is produced by transfection of vector plasmid constructions into the helper-free packaging cell line PA317. Before infection of patients' cells, the vector will have been shown to be free of helper virus. In the event that helper virus is detected, the vector will be produced in the GP+envAM12 packaging cell line in which the viral gag and pol genes are separated from the env, further reducing the likelihood of helper virus production.
3) Transduction Protocol
The cultured primary fibroblasts will be incubated with supernatant from the packaging cell line as described (20). Supernatant from these cells will be tested for adventitious agents and replication competent virus as described (16) and outlined in Table 1. The fibroblasts are washed and then grown in culture media containing G418, (a neomycin analogue) to select for transduced cells expressing the NeoR gene. The G418-resistant cells will be tested for expression of the IL-2 gene by measuring the concentration of IL-2 in the culture supernatant by an enzyme linked immunosorbent assay (ELISA) (12). G418-resilient cells expressing IL-2 will be stored at -70° C. until required for subsequent use in immunizations.
              TABLE 1                                                     
______________________________________                                    
Adventitious Agents and Safety Testing                                    
______________________________________                                    
           1.  Sterility                                                  
           2.  Mycoplasma                                                 
           3.  General Safety                                             
           4.  Viral Testing                                              
               LCM Virus                                                  
               Thymic agent                                               
               S+/L- eco                                                  
               S+/L- xeno                                                 
               S+/L- ampho                                                
               3T3 amplification                                          
               MRC-5/Vero                                                 
______________________________________
4) Preparation of Irradiated Tumor Cells
Tumors obtained form clinically indicated surgical resections or from superficial lymph node or skin metastases will be minced into 2-3 mm pieces and treated with collagenase and DNAse to facilitate separation of the tumor into a single cell suspension. The collected cells will be centrifuged and washed in RPMI 1640 media and then cryopreserved in a solution containing 10% dimethyl sulphoxide and 50% fetal calf serum in RPMI 1640 media. The cells will be stored in liquid nitrogen until the time of administration. Prior to their use in subcutaneous immunizations, the cells will be thawed, washed in media free of immunogenic contaminants, and irradiated with 4,000 rads per minute for a total of 20,000 rads in a cesium irradiator.
5) Patient Selection
Patients will have a histologically confirmed diagnosis of cancer. Patients with tumors that must be resected for therapeutic purposes or with tumors readily accessible for biopsy are most appropriate for this embodiment of the invention.
6) Pretreatment Evaluation
The following pretreatment evaluations will be performed:
1) History and physical examination including a description and quantification of disease activity.
2) Performance Status Assessment
0=Normal, no symptoms
1=Restricted, but ambulatory
2=Up greater than 50% of waking hours, capable of self-care
3=Greater than 50% of waking hours confined to bed or chair, limited self-care
4=Bedridden
3) Pretreatment Laboratory:
CBC with differential, platelet count, PT, PTT, glucose, BUN, creatinine, electrolytes, SGOT, SGPT, LDH, alkaline phosphatase, bilirubin, uric acid, calcium, total protein albumin.
4) Other Analyses:
Urinalysis
CH50, C3 and C4 serum complement levels Immunophenotyping of peripheral blood B cell and T cell subsets
Assays for detectable replication-competent virus in peripheral blood cells
PCR assays of peripheral blood leukocytes for NeoR, IL-2 and viral env
5) Other Pretreatment Evaluation:
Chest X-ray and other diagnostic studies including computerized tomography (CT), magnetic resonance imaging (MRI) or radionuclide scans may be performed to document and quantify the extent of disease activity.
Follow-up evaluations of these assessments at regular intervals during the course of therapy (approximately every 1 to 3 months) will be useful in determining response to therapy and potential toxicity, permitting adjustments in the number of immunizations administered.
7) Restrictions on Concurrent Therapy
For optimal effects of this treatment, patients should receive no concurrent therapy which is known to suppress the immune system.
8) Final Formulation
Each patient will receive subcutaneous immunizations with a mixture if irradiated tumor cells and autologous fibroblast CE cells genetically modified to secrete IL-2. Approximately 107 tumor cells will be mixed with 107 fibroblasts known to secrete at least 20 units/ml of IL-2 in tissue culture when semi-confluent (12). The irradiated tumor cells and genetically modified fibroblasts will be placed in a final volume of 0.2 ml normal saline for immunization.
9) Dose Adjustments
At least two subcutaneous immunizations will be administered, two weeks apart, with irradiated tumor cells and autologous fibroblasts genetically modified to secrete IL-2. If no toxicity is observed, subsequent booster immunizations may be administered periodically (at least one week apart) to optimize the anti-tumor immune response.
J) Treatment of Potential Toxicity
Toxic side effects are not expected to result from these immunizations. However, potential side effects of these immunizations are treatable in the following manner:
If massive tumor cell lysis results, any resulting uric acid nephropathy, adult respiratory distress syndrome, disseminated intravascular coagulation or hyperkalemia will be treated using standard methods.
Local toxicity at the sites of immunization will be treated with either topical steroids and/or surgical excision of the injection site as deemed appropriate.
Hypersensitivity reactions such as chills, fever and/or rash will be treated symptomatically with antipyretics and antihistamines. Patients should not be treated prophylactically. Should arthralgias, lymphadenopathy or renal dysfunction occur, treatment with corticosteroids and/or antihistamines will be instituted. Anaphylaxis will be treated by standard means such as administration of epinephrine, fluids, and steroids.
EXAMPLE II A. Retroviral IL-2 Gene Transfer and Expression in Fibroblasts
Retroviral vectors were employed to transfer and express IL-2 and neomycin phosphotransferase genes in murine and primary human fibroblasts. The retroviral vector DC/TKIL2 produced by Gilboa and co-workers (Gansbacher, et al., J. Exp. Med. 172:1217-1223, 1990, which is incorporated herein by reference) was utilized to transduce murine fibroblasts for application in an animal tumor model (see Section B below). Human fibroblasts were transduced with the retroviral vector LXSN-RI-IL2 (SEQ ID NO:1). Schematic diagrams of the structure of these retroviral vectors are provided in FIG. 1. A more complete description of the LXSN-RI-IL2 vector, including its nucleotide sequence, is provided in Example III and in Tables 2, 3, 4, 5 and 6 (SEQ ID NO:1).
Following infection with the described vectors and selection for 2-3 weeks in growth media containing the neomycin analogue G418, Balb/c and human embryonic fibroblast culture supernatants were harvested and tested for IL-2 by an enzyme-linked immunosorbent assay (ELISA). FIG. 2 depicts the levels of IL-2 secreted by the transduced fibroblasts.
These results can be confirmed using negative control fibroblasts infected with an N2-derived retroviral vector expressing an irrelevant gene such as luciferase or β-galactosidase and studies with adult human fibroblasts.
Biological activity of the IL-2 expressed by the transduced human fibroblasts was confirmed by a cell proliferation bioassay employing an IL-2 dependent T cell line. In this assay, supernatant from the transduced fibroblasts and control unmodified fibroblasts were incubated with the IL-2 dependent T cell line CTLL-2. Incorporation of 3 H-thymidine was measured as an indicator of cell proliferation and IL-2 activity (FIG. 3).
B. Efficacy of Transduced Fibroblasts in an Animal Tumor Model
The efficacy of fibroblasts genetically modified to secrete IL-2 was tested in an animal model of colorectal carcinoma. In these studies, the Balb/c CT26 tumor cell line was injected subcutaneously with Balb/c fibroblasts transduced to express IL-2. Control groups included animals injected with 1) a mixture of CT26 tumor cells and unmodified fibroblasts; 2) CT26 tumor cells without fibroblasts and 3) transduced fibroblasts alone. No tumors were detected in 3/8 animals treated with transduced fibroblasts and CT26 cells. In contrast, all untreated control animals (8/8) injected with CT26 tumor cells developed palpable tumors. No tumors were detected in the animals inoculated with transduced fibroblasts without CT26 tumor cells. The mean CT26 tumor size in Balb/c mice injected with the IL-2 secreting fibroblasts was considerably smaller compared to the control groups (FIG. 4). A multivariate non-parametric statistical procedure (Koziol, et al., Biometries 37:383-390, 1981 and Koziol, et al., Computer Prog. Biomed. 19:69-74, 1984, which is incorporated herein by reference) was utilized to evaluate differences in tumor growth among the treatment groups. The tumor growth curves for the four treatment groups presented in FIG. 4 were significantly different (p=0.048). Subsequent comparisons between treatment groups revealed a significant difference (p<0.05) in tumor growth between animals injected with CT26 tumor cells alone and animals treated with 2×106 transduced fibroblasts and CT26 tumor cells (FIG. 4).
EXAMPLE III A. Project Overview
Lymphokine gene therapy of cancer will be evaluated in cancer patients who have failed conventional therapy. An N2-derived vector containing the neomycin phosphotransferase gene will be used. This vector has been employed by a number of investigators for in vitro and in vivo studies including recently approved investigations with human subjects (Rosenberg et al., N. Eng. J. Med., 323:570-578, 1990). The lymphokine vectors used in this investigation will be generated from the N2-derived vector, LXSN, developed and described by Miller et al., Mol. Cell Biol. 6:2895, 1986 and Miller et al., BioTechniques 7:980, 1989, which are incorporated herein by reference. The vector LXSN-RI-IL2 contains human IL-2 cDNA under the control of the retroviral 5' LTR promoter and the neomycin phosphotransferase gene under the control of the SV40 promoter (see FIG. 1). The normal human IL-2 leader sequence has been replaced with a chimeric sequence containing rat insulin and human IL-2 leader sequences (see Tables 2, 3, 4, 5 and 6 (SEQ ID NO:1)). This chimeric leader sequence enhances IL-2 gene expression.
To construct the LXSN-RI-IL2 vector, the bacterial plasmid pBC12/CMV/IL2 (Cullen, B. R., DNA 7:645-650, 1988, which is incorporated herein by reference) containing the full-length IL-2 cDNA and chimeric leader sequence was digested with HindIII and the ends were blunted using Klenow polymerase. IL-2 cDNA was subsequently released from the plasmid by digestion with BamHI. The IL-2 fragment was purified by electrophoresis in a 1% agarose gel and the appropriate band was extracted utilizing a glass powder method. Briefly, the gel slice was dissolved in 4M NaI at 55°. After cooling to room temperature, 4 μl of oxidized silica solution (BIO-101, La Jolla, Calif.) was added to adsorb the DNA. The silica was then washed with a cold solution of 50% ethanol containing 0.1M NaCl in TE buffer. The DNA was eluted from the silica by heating at 55° in distilled H2 O. The purified IL-2 cDNA was then directionally ligated into the HpaI-BamHI cloning sites of the pLXSN vector. A more complete description of the pLXSN-RI-IL2 vector and its partial nucleotide sequence are provided in Tables 2, 3, 4, 5 and 6 (SEQ ID NO:1).
              TABLE 2                                                     
______________________________________                                    
Description of the LXSN-RI-IL2                                            
from position 1 to 6365                                                   
Bases      Description                                                    
______________________________________                                    
   1-589   Moloney murine sarcoma virus 5' LTR                            
 659-1458  The sequence of the extended packaging                         
           signal                                                         
1469-2151  IL-2 cDNA with chimeric leader sequencc                        
1469-1718  IL-2 chimeric leader sequence                                  
1647-1718  coding region of the signal peptide                            
1719-2151  Mature IL-2 coding sequence                                    
2158-2159  Mo mu sarcoma virus end/SV 40 start                            
2159-2503  Simian virus 40 early promoter                                 
2521-2522  Simian virus DNA end/Tn5 DNA start                             
2557-3351  Neomycin phosphotransferase                                    
3370-3371  Tn5 DNA end/Moloney murine leukemia                            
           virus start                                                    
3411-4004  Moloney murine leukemia virus 3' LTR                           
4073-4074  Moloney murine leukemia DNA end/pBR322                         
           DNA start                                                      
4074-6365  Plasmid backbone                                               
______________________________________                                    

                                  TABLE 3                                 
__________________________________________________________________________
Enzyme                                                                    
      (# Cuts)                                                            
          Position(s)                                                     
__________________________________________________________________________
Aat1  (2) 1961, 2481                                                      
Aat2  (2) 811, 6295                                                       
Acc1  (1) 4252                                                            
Acc2  (19)                                                                
          392, 394, 445, 969, 971, 1193, 2751, 3052, 3084, 3807,          
          3809, 4081, 4083. 4186, 4527, 5108, 5438, 5931, 6263            
Acy1  (5) 808, 2685, 3860, 5910, 6292                                     
Afl1  (13)                                                                
          260, 273, 328, 626, 756, 1277, 3201, 3676, 3689, 3744,          
          4041, 5511, 5733                                                
Afl2  (4) 34, 1064, 1955, 3446                                            
Afl3  (2) 1592, 4480                                                      
Aha1  (20)                                                                
          161, 237, 473, 474, 602, 644, 789, 2689, 2849, 3578,            
          3653, 3888, 3889, 4017, 4059, 4126, 4161, 4860, 5556, 5907      
Aha2  (5) 808, 2685, 3860, 5910, 6292                                     
Aha3  (3) 5239, 5258, 5950                                                
Alu1  (33)                                                                
          29, 33, 119, 190, 411, 654, 734, 742, 1470, 1486,               
          1751, 1935, 2003, 2446, 2500, 2791, 3249, 3441, 3445, 3532,     
          3607, 3826,                                                     
          4069, 4122, 4141, 4422, 4648, 4738, 4784, 5041, 5562, 5662,     
          5725                                                            
Alw1  (20)                                                                
          1110, 1414, 1665, 2018, 2147, 2160, 2529, 2553, 2864, 2929,     
          3110, 4027, 5041, 5127, 5129, 5225, 5226, 5689, 6006, 6010      
AlwN1 (4) 231, 3572, 3647, 4896                                           
Aoc1  (2) 847, 1076                                                       
Aoc2  (19)                                                                
          323, 413, 426, 597, 1583, 1721, 2631, 2724, 2798, 2988,         
          3050, 3739, 3828, 3841, 4012, 4300, 4798, 5959, 6044            
Aos1  (2) 2787, 5595                                                      
ApaL1 (4) 1717, 4296, 4794, 6040                                          
Apy1  (22)                                                                
          315, 623, 801, 814, 1227, 1252, 1275, 1295, 1325, 1526,         
          1536, 1558, 1630, 2196, 2251, 2268, 3072, 3731, 4038, 4508,     
          4629, 4642                                                      
Aqu1  (6) 241, 472, 1998, 3821, 3854, 3887                                
Ase1  (2) 1801, 5545                                                      
Asp700                                                                    
      (1) 5972                                                            
Asp7l8                                                                    
      (2) 476, 3891                                                       
AspA1 (1) 1145                                                            
Asu1  (29)                                                                
          169, 200, 245, 260, 273, 328, 626, 756, 826, 839,               
          1043, 1254, 1277, 1532, 1649, 3201, 3541, 3586, 3616, 3661,     
          3676, 3689,                                                     
          3744, 4041, 5415, 5494, 5511, 5733, 6349                        
Ava1  (6) 241, 472, 1998, 3821, 3854, 3887                                
Ava2  (13)                                                                
          260, 273, 328, 626, 756, 1277, 3201, 3676, 3689, 3744,          
          4041, 5511, 5733                                                
Ava3  (2) 2232, 2304                                                      
Avr2  (2) 1962, 2482                                                      
Bal1  (3) 658, 1169, 2767                                                 
BamH1 (1) 2152                                                            
Ban1  (9) 318, 476, 1200. 2684, 2719, 3734, 3859, 3891, 5321              
Ban2  (8) 413, 426, 597, 1583, 3050, 3828, 3841, 4012                     
Bbe1  (2) 2688, 3863                                                      
Bbv1  (22)                                                                
          969, 997, 1738, 2493, 2632, 2758, 2800, 2816, 2909, 3321,       
          4060, 4131, 4228, 4372, 4390, 4809, 4899, 4902, 5108, 5411,     
          5600, 5802                                                      
Bcl1  (1) 2526                                                            
Bgl1  (2) 2435, 5493                                                      
Bsp1286I                                                                  
      (19)                                                                
          323, 413, 426, 597, 1583, 1721, 2631, 2724, 2798, 2988,         
          3050, 3739, 3828, 3841, 4012, 4300, 4798, 5959, 6044            
BspH1 (3) 5200, 6208, 6313                                                
BspM1 (4) 150l, 2500, 2572, 2953                                          
BssH2 (4) 392, 443, 3082, 3807                                            
BstE2 (1) 1145                                                            
BstN1 (22)                                                                
          315, 623, 801, 814, 1227, 1252, 1275, 1295, 1325, 1526,         
          1536, 1558, 1630, 2196, 225l, 2268, 3072, 3731, 4038, 4508,     
          4629, 4642                                                      
BstU1 (19)                                                                
          392, 394, 445, 969, 971, 1193, 2751, 3052, 3084, 3807,          
          3809, 4081, 4083, 4186, 4527, 5108, 5438, 5931, 6263            
BstX1 (1) 2060                                                            
BstY1 (ll)                                                                
          2010, 2152, 2521, 2856, 3102, 5121, 5132, 5218, 5230, 5998,     
          6015                                                            
Bsu36I                                                                    
      (2) 847, 1076                                                       
Ccr1  (1) 1998                                                            
Cfo1  (31)                                                                
          394, 396, 445, 447, 714, 971, 2679, 2687, 2751, 2788,           
          3054, 3084, 3086, 3314, 3809, 3811, 3862, 4083, 4186, 4216,     
          4357, 4390,                                                     
          4660, 4727, 4827, 5001, 5110, 5503, 5596, 5933, 6265            
Cfr1  (9) 656, 790, 1167, 1188, 2591, 2765, 3156, 3183, 5761              
Cfr10I                                                                    
      (3) 3004, 3185, 5453                                                
Cfr13X                                                                    
      (29)                                                                
          169, 200, 245, 260, 273, 328, 626, 756, 826, 839,               
          1043, 1254, 1277, 1532, 1649, 3201, 3541, 3586, 3616, 3661,     
          3676, 3689,                                                     
          3744, 4041, 5415, 5494, 5511, 5733, 6349                        
Cvn1  (2) 847, 1076                                                       
Dde1  (23)                                                                
          75, 165, 191, 282, 553, 847, 1076, 1348, 1692, 2442,            
          3348, 3487, 3582, 3657, 3698, 3879, 3967, 4290, 4755, 5164,     
          5330, 5870,                                                     
          6296                                                            
Dpn1  (30)                                                                
          95, 1104, 1236, 1421, 1659, 2012, 2154, 2523, 2528, 2547,       
          2858, 2936, 3017, 3026, 3104, 3507, 4021, 5048, 5123, 5134,     
          5142, 5220,                                                     
          5232, 5337, 5678, 5696, 5742, 6000, 6017, 6053                  
Dra1  (3) 5239, 5258, 5950                                                
Dra2  (4) 328, 1277, 3744, 6349                                           
Eae1  (9) 656, 790, 1167, 1108, 2591, 2765, 3156, 3183, 5761              
Eag1  (2) 790, 2591                                                       
Eco47I                                                                    
      (13)                                                                
          260, 273, 328, 626, 756, 1277, 3201, 3676, 3689, 3744,          
          4041, 5511, 5733                                                
Eco52I                                                                    
      (2) 790, 259l                                                       
Eco81X                                                                    
      (2) 847, 1076                                                       
EcoN1 (2) 850, 1450                                                       
Eco0109I                                                                  
      (4) 328, 1277, 3744, 6349                                           
EcoR1 (1) 1460                                                            
EcoR1*                                                                    
      (14)                                                                
          938, 1037, 1460, 1798, 1805, 1928, 2064, 2121, 2236, 2308,      
          2400, 5240, 5546, 5801                                          
EcoR2 (22)                                                                
          313, 621, 799, 812, 1225, 1250, 1273, 1293, 1323, 1524,         
          1534, 1556, 1628, 2194, 2249, 2266, 3070, 3729, 4036, 4506,     
          4627, 4640                                                      
EcoR5 (4) 137, 213, 3554, 3629                                            
EcoT22I                                                                   
      (2) 2232, 2304                                                      
Fdi2  (2) 2787, 5595                                                      
Fnu4H1                                                                    
      (41)                                                                
          793, 967, 983, 986, 1191, 1752, 2430, 2507, 2594, 2646,         
          2657, 2747, 2752, 2789, 2830, 2917, 2920, 2923, 3159, 3255,     
          3296, 3310,                                                     
          4074, 4120, 4217, 4270, 4386, 4404, 4407, 4525, 4680, 4823,     
          4888, 4891,                                                     
          5097, 5425, 5614, 5764, 5791, 5886, 6115                        
FnuD2 (9) 392, 394, 445, 969, 971, 1193, 2751, 3052, 3084, 3807,          
          3809, 4081, 4083, 4186, 4527, 5108, 5438, 5931, 6263            
Fok1  (13)                                                                
          498, 1198, 1358, 1679, 2333, 2552, 3009, 3034, 3912, 4168,      
          5339, 5520, 5807                                                
Fsp1  (2) 2787, 5595                                                      
Hae2  (4) 2688, 3863, 4358, 4728                                          
Hae3  (35)                                                                
          171, 202, 247, 658, 792, 828, 840, 1045, 1169, 1190,            
          1255, 1534, 1650, 1866, 1961, 2423, 2429, 2438, 2481, 2593,     
          2767, 3158,                                                     
          3185, 3543, 3588, 3618, 3663, 4495, 4506, 4524, 4958, 5416,     
          5496, 5763,                                                     
          6350                                                            
Hap2  (30)                                                                
          161, 237, 473, 601, 643, 789, 2590, 2667, 2689, 2717,           
          2848, 2938, 3005, 3186, 3578, 3653, 3888, 4016, 4058, 4126,     
          4160, 4687,                                                     
          4834, 4860, 5050, 5454, 5488, 5555, 5665, 5907                  
Hga1  (8) 455, 707, 960, 1580, 4175, 4591, 5169, 5899                     
HgiA1 (9) 413, 1721, 2798, 2988, 3828, 4300, 4798, 5959, 6044             
Hha1  (31)                                                                
          394, 396, 445, 447, 714, 971, 2679, 2687, 2751 2788,            
          3054, 3084, 3086, 3314, 3809, 3811, 3862, 4083, 4186, 4216,     
          4357, 4390,                                                     
          4660, 4727, 4827, 5001, 5110, 5503, 5596, 5933, 6265            
HinP1 (31)                                                                
          392, 394, 443, 445, 712, 969, 2677, 2685, 2749, 2786,           
          3052, 3082, 3084, 3312, 3807, 3809, 3860, 4081, 4184, 4214,     
          4355, 4388,                                                     
          4658, 4725, 4825, 4999, 5108, 5501, 5594, 5931, 6263            
Hinc2 (1) 5914                                                            
Hind2 (1) 5914                                                            
Hind3 (1) 2498                                                            
Hinf1 (14)                                                                
          298, 517, 857, 868, 1553, 1814, 3170, 3304, 3356, 3881,         
          4380, 4455, 4851, 5368                                          
Hpa2  (30)                                                                
          161, 237, 473, 601, 643, 789, 2590, 2667, 2689, 2717,           
          2848, 2938, 3005, 3186, 3578, 3653, 3888, 4016, 4058, 4126,     
          4160, 4687,                                                     
          4834, 4860, 5050, 5454, 5488, 5555, 5665, 5907                  
Hph1  (11)                                                                
          1214, 1240, l817, 2863, 4102, 4111, 5216, 5443, 5859, 6065,     
          6100                                                            
Kpn1  (2) 480, 3895                                                       
Mae1  (15)                                                                
          30, 293, 689, 727, 739, 1452, 1606, 1893, 1963, 2483,           
          3442, 3709, 4975, 5228, 5563                                    
Mae2  (11)                                                                
          808, 1139, 1180, 1987, 2801, 2988, 4233, 5183, 5599, 5972,      
          6292                                                            
Mae3  (20)                                                                
          38, 1052, 1080, 1145, 1289, 1478, 1706, 2805, 3111, 3450,       
          4134, 4229, 4836, 4899, 5015, 5298, 5629, 5687, 5840, 6028      
Mbo1  (30)                                                                
          93, 1102, 1234, 1419, 1657, 2010, 2152, 2521, 2526, 2545,       
          2856, 2934, 3015, 3024, 3102, 3505, 4019, 5046, 5121, 5132,     
          5140, 5218,                                                     
          5230, 5335, 5676, 5694, 5740, 5998, 6015, 6051                  
Mbo2  (17)                                                                
          444, 1145, 1356, 1575, 1617, 1908, 1911, 3046, 3256, 3336,      
          4351, 5142, 5213, 5968, 6046, 6155, 6351                        
Mn11  (54)                                                                
          291, 444, 508, 534, 560, 639, 841, 939, 1227, l330,             
          1363, 1369, 1372, 1378, 1408, 1411, 1426, 1433, 1449, 1559,     
          1620, 1909,                                                     
          1921, 2412, 2418, 2443, 2449, 2455, 2458, 2470, 2508, 2535,     
          2599, 2735,                                                     
          3092, 3286, 3707, 3859, 3878, 3923, 3948, 3974, 4054, 4087,     
          4117, 4379,                                                     
          4587, 4662, 4911, 5311, 5392, 5540, 5746, 6339                  
Mse1  (22)                                                                
          35, 1065, 1177, 1207, 1231, 1801, 1843, 1956, 1971, 2124,       
          2139, 3447, 4261, 5186, 5238, 5243, 5257, 5310, 5545, 5584,     
          5949, 6321                                                      
Msp1  (30)                                                                
          161, 237, 473, 601, 643, 789, 2590, 2667, 2689, 2717,           
          2848, 2938, 3005, 3186, 3578, 3653, 3888, 4016, 4058, 4126,     
          4160, 4687,                                                     
          4834, 4860, 5050, 5454, 5488, 5555, 5665, 5907                  
Mst1  (2) 2787, 5595                                                      
Mst2  (2) 847, 1076                                                       
Mva1  (22)                                                                
          315, 623, 801, 814, 1227, 1252, 1275, 1295, 1325, 1526,         
          1536, 1558, 1630, 2196, 2251, 2268, 3072, 3731, 4038, 4508,     
          4629, 4642                                                      
Nae1  (1) 3187                                                            
Nar1  (2) 2685, 3860                                                      
Nci1  (20)                                                                
          161, 237, 473, 474, 602, 644, 789, 2689, 2849, 3578,            
          3653, 3888, 3889, 4017, 4059, 4126, 4161, 4860, 5556, 5907      
Nco1  (2) 2389, 3117                                                      
Nde1  (1) 4303                                                            
Nde2  (30)                                                                
          93, 1102, 1234, 1419, 1657, 2010, 2152, 2521, 2526, 2545,       
          2856, 2934, 3015, 3024, 3102, 3505, 4019, 5046, 5121, 5132,     
          5140, 5218,                                                     
          5230, 5335, 5676, 5694, 5740, 5998, 6015, 6051                  
Nhe1  (3) 29, 1605, 3441                                                  
Nla3  (26)                                                                
          61, 1263, 1596, 1649, 1835, 1856, 2030, 2230, 2302, 2393,       
          2559, 2904, 3090, 3121, 3147, 3473, 4119, 4224, 4484, 5204,     
          5695, 5705,                                                     
          5783, 5819, 6212, 6317                                          
Nla4  (28)                                                                
          153, 246, 262, 320, 478, 627, 758, 827, 959, 1202,              
          1279, 2154, 2200, 2272, 2686, 2721, 3678, 3736, 3861, 3893,     
          4042, 4512,                                                     
          4551, 5323, 5417, 5458, 5669, 6259                              
Nsi1  (2) 2232, 2304                                                      
Nsp(7524)1                                                                
      (8) 1596, 1835, 1856, 2230, 2302, 3090, 4119, 4484                  
Nsp(7524)2                                                                
      (19)                                                                
          323, 413, 426, 597, 1583, 1721, 2631, 2724, 2798, 2988,         
          3050, 3739, 3828, 3841, 4012, 4300, 4798, 5959, 6044            
NspB2 (12)                                                                
          119, 190, 1751, 2158, 2791, 3532, 3607, 3989, 4192, 4822,       
          5067, 6008                                                      
NspH1 (8) 1596, 1835, 1856, 2230, 2302, 3090, 4119, 4484                  
PaeR7I                                                                    
      (1) 1998                                                            
Pal1  (35)                                                                
          171, 202, 247, 658, 792, 828, 840, 1045, 1169, 1190,            
          1255, 1534, 1650, 1866, 1961, 2423, 2429, 2438, 2481, 2593,     
          2767, 3158,                                                     
          3185, 3543, 3588, 3618, 3663, 4495, 4506, 4524, 4958, 5416,     
          5496, 5763,                                                     
          6350                                                            
Ple1  (7) 865, 1547, 3350, 3889, 4374, 4859, 5362                         
PpuM1 (3) 328, 1277, 3744                                                 
Pss1  (4) 331, 1280, 3747, 6352                                           
Pst1  (6) 987, 1163, 1888, 2511, 2738, 5618                               
Pvu1  (1) 5743                                                            
Pvu2  (6) 119, 190, 1751, 2791, 3532, 3607                                
Rsa1  (10)                                                                
          347, 478, 725, 1342, 1519, 1597, 2991, 3893, 4288, 5853         
Rer2  (1) 3201                                                            
Sac1  (2) 413, 3828                                                       
Sau1  (2) 847, 1076                                                       
Sau3A1                                                                    
      (30)                                                                
          93, 1102, 1234, 1419, 1657, 2010, 2152, 2521, 2526, 2545,       
          2856, 2934, 3015, 3024, 3102, 3505, 4019, 5046, 5121, 5132,     
          5140, 5218,                                                     
          5230, 5335, 5676, 5694, 5740, 5998, 6015, 6051                  
Sau96I                                                                    
      (29)                                                                
          169, 200, 245, 260, 273, 328, 626, 756, 826, 839,               
          1043, 1254, 1277, 1532, 1649, 3201, 3541, 3586, 3616, 3661,     
          3676, 3689,                                                     
          3744, 4041, 5415, 5494, 5511, 5733, 6349                        
Sca1  (1) 5853                                                            
ScrF1 (42)                                                                
          161, 237, 315, 473, 474, 602, 623, 644, 789, 801,               
          814, 1227, 1252, 1275, 1295, 1325, 1526, 1536, 1558, 1630,      
          2196, 2251,                                                     
          2268, 2689, 2849, 3072, 3578, 3653, 3731, 3888, 3889, 4017,     
          4038, 4059,                                                     
          4126, 4161, 4508, 4629, 4642, 4860, 5556, 5907                  
Sdu1  (19)                                                                
          323, 413, 426, 597, l583, 1721, 2631, 2724, 2798, 2988,         
          3050, 3739, 3828, 3841, 4012, 4300, 4798, 5959, 6044            
Sec1  (38)                                                                
          159, 235, 314, 324, 472, 536, 621, 622, 760, 799,               
          800, 812, 813, 1225, 1294, 1303, 1323, 1324, 1525, 1557, 1962,  
          2194,                                                           
          2266, 2389, 2424, 2433, 2482, 2848, 3117, 3576, 3651, 3730,     
          3740, 3887,                                                     
          3950, 4036, 4037, 4640                                          
SfaN1 (23)                                                                
          258, 520, 997, 1657, 2107, 2239, 2311, 2643, 2898, 2984,        
          3048, 3114, 3323, 3674, 3934, 4146, 4281, 4317, 4357, 4577,     
          5629, 5820,                                                     
          6069                                                            
Sfi1  (1) 2435                                                            
Sma1  (2) 474, 3889                                                       
Spe1  (1) 726                                                             
Sph1  (4) 1835, 2230, 2302, 3090                                          
Ssp1  (1) 6177                                                            
Sst1  (2) 413, 3828                                                       
Stu1  (2) 1961, 2481                                                      
Sty1  (9) 324, 536, 1303, 1962, 2389, 2482, 3117, 3740, 3950              
Taq1  (15)                                                                
          860, 1096, 1407, 1418, 1660, 1999, 2514, 2798, 2954, 2978,      
          3014, 3176, 3367, 4580, 6024                                    
Tha1  (19)                                                                
          392, 394, 445, 969, 971, 1193, 2751, 3052, 3084, 3807,          
          3809, 4081, 4083, 4186, 4527, 5108, 5438, 5931, 6263            
Tth111I                                                                   
      (6) 465, 877, 1275, 2803, 3880, 4227                                
Xba1  (2) 1892, 3708                                                      
Xho1  (1) 1998                                                            
Xho2  (11)                                                                
          2010, 2152, 2521, 2856, 3102, 5121, 5132, 5218, 5230, 5998,     
          6015                                                            
Xma1  (2) 472, 3887                                                       
Xma3  (2) 790, 2591                                                       
Xmn1  (1) 5972                                                            
Xor2  (1) 5743                                                            
__________________________________________________________________________

              TABLE 4                                                     
______________________________________                                    
enzymes which do not cut LXSNRIIL2:                                       
______________________________________                                    
Acc3    Bg12     Cla1      Hpa1  Nru1   SnaB1                             
Apa1    Bsm1     Dra3      Mlu1  PflM1  Spl1                              
Asu2    BspM2    Eco47III  Mro1  Sac2   Sst2                              
Ban3    BstB1    Esp1      Not1  Sal1                                     
______________________________________                                    

TABLE 5                                                                    
  - From 1 to 6365. Numbered from position 1.                              
  ##STR1##                                                                 
  Aat1- - - - - - - - - - - + - - - - - - - - - - - 1 - - - - - 1 - - - -  
 - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - -   
 - + - - -                                                                 
  -                                                               Aat2- -  
 - - - - - - - 1 - + - - - - - - - - - - - + - - - - - - - - - - - + - -   
 - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - -   
 1 -                                                                       
  Acc1- - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - -  
 - + - - - - - - - - - - - + - - 1 - - - - - - - - + - - - - - - - - - -   
 - + - - -                                                                 
  -                                                               Acc2- -  
 - - 2 1 - - - - - 2 - - 1 - - - - - - - - + - - - - - - - - 1 - - + 2 -   
 - - - - - - - 2 - + 2 1 - - - 1 - - - - - + 1 - - - 1 - - - - - 1 + - -   
 1 -                                                                       
  Acy1- - - - - - - - - 1 - + - - - - - - - - - - - + - - - - - - - 1 - -  
 - + - - - - - - - - - 1 - + - - - - - - - - - - - + - - - - - - - - - -   
 1 + - -                                                                   
  1 -                                                               Afl1-  
 - - 3 - - - 1 1 - - + - - - 1 - - - - - - - + - - - - - - - - - - - + -   
 - 1 - - - - 2 1 - - + 1 - - - - - - - - - - + - - - - - 1 - - 1 - - + -   
 - -                                                                       
  -                                                                        
 Afl21 - - - - - - - - - - + 1 - - - - - - - - - - 1 - - - - - - - - - -   
 - + - - - - - 1 - - - - - + - - - - - - - - - - - + - - - - - - - - - -   
 - + - - -                                                                 
  -                                                               Afl3- -  
 - - - - - - - - - + - - - - - - 1 - - - - + - - - - - - - - - - - + - -   
 - - - - - - - - - + - - - - - 1 - - - - - + - - - - - - - - - - - + - -   
 -                                                                         
  -                                                                       A
 ha1- 1 1 - - 2 - 2 - 1 - + - - - - - - - - - - - + - - - - - - - - 1 1 -  
 + - - - - - - 1 1 - - 2 1 1 2 - - - - - - - 1 - + - - - - - - 1 - - - 1   
 + - - -                                                                   
  -                                                                 Aha2-  
 - - - - - - - - 1 - + - - - - - - - - - - - + - - - - - - - 1 - - - + -   
 - - - - - - - - 1 - + - - - - - - - - - - - + - - - - - - - - - - 1 + -   
 -                                                                         
  1 -                                                                     A
 ha3- - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - -  
 + - - - - - - - - - - - + - - - - - - - - - - - + - - 2 - - - - - - - 1+  
 - - -                                                                     
  -                                                                       2
 Alu1 1 1 - 1 - - 1 2 - - + - - - - - 2 - - 1 - - 2 - - - - - 2 - - - 1 -  
 + - - 1 - 1 1 2 - - 1 - + 1 2 - - 1 - - 1 2 - - + 1 - - - - - 1 1 1 - -   
 + - - -                                                                   
  -                                                                 Alw1-  
 - - - - - - - - - - + - 1 - - 1 - - 1 - - - + 1 2 - - - - 2 - - - 2 + -   
 1 - - - - - - - - - 1 - - - - - - - - - - - + 1 2 2 - - - - 1 - - - 2 -   
 - -                                                                       
  -                                                                        
 AlwN1- - 1 - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - -  
 - + - - - - - - 1 1 - - - + - - - - - - - - - - 1 + - - - - - - - - - -   
 - + - - -                                                                 
  -                                                               Aoc1- -  
 - - - - - - - - 1 + 1 - - - - - - - - - - + - - - - - - - - - - - + - -   
 - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - -   
 -                                                                         
  -                                                                       A
 oc2- - - 1 1 1 - 1 - - - + - - - - - - 1 - 1 - - + - - - - - - - 1 1 1 -  
 1 1 - - - - - - - 1 2 - 1 - - - 1 - - - - - 1 - + - - - - - - - - - - 1   
 1 - - -                                                                   
  -                                                                 Aos1-  
 - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - 1 - + -   
 - - - - - - - - - - + - - - - - - - - - - - + - - - - - - 1 - - - - + -   
 - -                                                                       
  -                                                                        
 ApaLi- - - - - - - - - - - + - - - - - - - - 1 - - + - - - - - - - - - -  
 - + - - - - - - - - - - - + - - - 1 - - - - - 1 - + - - - - - - - - - -   
 - 1 - - -                                                                 
  -                                                               Apy1- -  
 - 1 - - - 1 - 2 - + - - 2 3 - - 3 1 - - - + - - 3 - - - - - - - - + 1 -   
 - - - - - - 1 - - + 1 - - - - 1 - 2 - - - + - - - - - - - - - - - + - -   
 -                                                                         
  -                                                                       A
 qu1- - 1 - - 1 - - - - - + - - - - - - - - - - - 1 - - - - - - - - - - -  
 + - - - - - - - - - 2 1 + - - - - - - - - - - - + - - - - - - - - - - -   
 + - - -                                                                   
  -                                                                 Ase1-  
 - - - - - - - - - - + - - - - - - - - - 1 - + - - - - - - - - - - - + -   
 - - - - - - - - - - + - - - - - - - - - - - + - - - - - - 1 - - - - + -   
 - -                                                                       
  -                                                                        
 Asp700- - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - -   
 - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - -   
 - - 1 - - -                                                               
  -                                                             Asp718- -  
 - - - 1 - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - -   
 - - - - - - - - 1 + - - - - - - - - - - - + - - - - - - - - - - - + - -   
 -                                                                         
  -                                                                       A
 spA1- - - - - - - - - - - + - 1 - - - - - - - - - + - - - - - - - - - -   
 - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - -   
 - + - - -                                                                 
  -                                                               Asu1- - 3
  3 - - - 1 1 2 - + 1 - 1 1 - - 1 1 - - - + - - - - - - - - - - - + - - 1  
 - - - 2 4 1 - - + 1 - - - - - - - - - - + - - - - 1 2 - - 1 - - + - - -   
 1                                                                         
  Ava1- - 1 - - 1 - - - - - + - - - - - - - - - - - 1 - - - - - - - - - -  
 - + - - - - - - - - - 2 1 + - - - - - - - - - - - + - - - - - - - - - -   
 - + - - -                                                                 
  -                                                               Ava2- -  
 - 3 - - - 1 1 - - + - - - 1 - - - - - - - + - - - - - - - - - - - + - -   
 1 - - - - 2 1 - - + 1 - - - - - - - - - - + - - - - - 1 - - 1 - - + - -   
 -                                                                         
  -                                                                       A
 va3- - - - - - - - - - - + - - - - - - - - - - - + - - 1 1 - - - - - - -  
 + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - -   
 + - - -                                                                   
  -                                                                 Avr2-  
 - - - - - - - - - - + - - - - - - - - - - - 1 - - - - - 1 - - - - - + -   
 - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + -   
 - -                                                                       
  -                                                                        
 Bal1- - - - - - - 1 - - - + - 1 - - - - - - - - - + - - - - - - - - 1 -   
 - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - -   
 - + - - -                                                                 
  -                                                               BamH1-   
 - - - - - - - - - - + - - - - - - - - - - - + - 1 - - - - - - - - - + -   
 - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + -   
 - -                                                                       
  -                                                                        
 Bbe1- - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - 1 - -   
 - + - - - - - - - - - 1 - + - - - - - - - - - - - + - - - - - - - - - -   
 - + - - -                                                                 
  -                                                               Bbvl- -  
 - - - - - - - - - 2 - - - - - - - - 1 - - + - - - - - 1 - 1 1 2 1 + - -   
 - 1 - - - - - - - + 1 1 1 - 2 - - - - 1 2 + 1 - - - 1 - 1 - - 1 - + - -   
 -                                                                         
  -                                                                       B
 cl1- - - - - - - - - - - + - - - - - - - - - - - + - - - - - - 1 - - - -  
 + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - -   
 + - - -                                                                   
  -                                                                 Bgl1-  
 - - - - - - - - - - + - - - - - - - - - - - + - - - - 1 - - - - - - + -   
 - - - - - - - - - - + - - - - - - - - - - - + - - - - - 1 - - - - - + -   
 - -                                                                       
  -                                                                        
 Bsp1286I- - - 1 1 1 - 1 - - - + - - - - - - 1 - 1 - - + - - - - - - - 1   
 1 1 - 1 1 - - - - - - - 1 2 - 1 - - - 1 - - - - - 1 - + - - - - - - - -   
 - - 1 1 - - -                                                             
  -                                                           BspH1- - -   
 - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - -   
 - - - - - - - - + - - - - - - - - - - - + - 1 - - - - - - - - - + - 1 -   
 1                                                                         
  BspM1- - - - - - - - - - - + - - - - - 1 - - - - - + - - - - - 1 1 - -   
 - - 1 - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - -   
 - - + - - -                                                               
  -                                                             BssH2- -   
 - - 1 1 - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + 1 -   
 - - - - - - - 1 - + - - - - - - - - - - - + - - - - - - - - - - - + - -   
 -                                                                         
  -                                                                       B
 stE2- - - - - - - - - - - + - 1 - - - - - - - - - + - - - - - - - - - -   
 - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - -   
 - + - - -                                                                 
  -                                                               BstN1-   
 - - 1 - - - 1 - 2 - + - - 2 3 - - 3 1 - - - + - - 3 - - - - - - - - + 1   
 - - - - - - - 1 - - + 1 - - - - 1 - 2 - - - + - - - - - - - - - - - + -   
 - -                                                                       
  -                                                                        
 BstU1- - - - 2 1 - - - - - 2 - - 1 - - - - - - - - + - - - - - - - - 1 -  
 - + 2 - - - - - - - - 2 - + 2 1 - - - 1 - - - - - + 1 - - - 1 - - - - -   
 1 + - -                                                                   
  1 -                                                                      
    BstX1- - - - - - - - - - - + - - - - - - - - - - - + 1 - - - - - - -   
 - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - -   
 - - - + - - -                                                             
  -                                                           BstY1- - -   
 - - - - - - - - + - - - - - - - - - - - 1 - 1 - - - - 1 - - 1 - + 1 - -   
 - - - - - - - - + - - - - - - - - - - - + 1 1 2 - - - - - - - - 2 - - -   
 -                                                                         
  Bsu36I- - - - - - - - - - 1 + 1 - - - - - - - - - - + - - - - - - - - -  
 - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - -   
 - - + - - -                                                               
  -                                                             Ccr1- - -  
 - - - - - - - - + - - - - - - - - - - - 1 - - - - - - - - - - - + - - -   
 - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - -   
 -                                                                         
  Cfo1- - - - 2 2 - - 1 - - 1 - - - - - - - - - - - + - - - - - - - 2 1 1  
 - + 3 - - 1 - - - - - 3 - + 1 1 1 1 1 - - 1 1 1 - 1 1 - - - - 1 1 - - -   
 1 + - -                                                                   
  1 -                                                               Cfr1-  
 - - - - - - 1 - 1 - + - 1 1 - - - - - - - - + - - - - - - 1 - 1 - - + -   
 2 - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - 1 - - + -   
 - -                                                                       
  -                                                                        
 Cfr10I- - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - -   
 - - 1 - 1 - - - - - - - - - + - - - - - - - - - - - + - - - - 1 - - - -   
 - - + - - -                                                               
  -                                                             Cfr13I- - 3
  3 - - - 1 1 2 - + 1 - 1 1 - - 1 1 - - - + - - - - - - - - - - - + - - 1  
 - - - 2 4 1 - - + 1 - - - - - - - - - - + - - - - 1 2 - - 1 - - + - - -   
 1                                                                         
  Cvn1- - - - - - - - - - 1 + 1 - - - - - - - - - - + - - - - - - - - - -  
 - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - -   
 - + - - -                                                                 
  -                                                               Dde11 1  
 1 1 - - 1 - - - 1 + 1 - - - 1 - - - 1 - - + - - - - - 1 - - - - - + - -   
 - 1 - 1 1 1 1 - 1 1 - - - 1 - - - - 1 - - + - 1 - 1 - - - - - 1 - + - -   
 1 -                                                                       
  Dpn1- 1 - - - - - - - - - + - 11 - 1 - - 1 - - - 1 - 1 - - - - 3 - - -  1
2                                                                          
 2 - - - - 1 - - - - - 1 - - - - - - - - - - - + 2 2 2 1 - - - 2 1 - - 2   
 1 - -                                                                     
  -                                                                        
 Dra1- - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - -   
 - + - - - - - - - - - - - + - - - - - - - - - - - + - - 2 - - - - - - -   
 1 + - - -                                                                 
  -                                                               Dra2- -  
 - 1 - - - - - - - + - - - 1 - - - - - - - + - - - - - - - - - - - + - -   
 - - - - - - 1 - - + - - - - - - - - - - - + - - - - - - - - - - - + - -   
 -                                                                         
  1                                                                       E
 ae1- - - - - - - 1 - 1 - + - 1 1 - - - - - - - - + - - - - - - 1 - 1 - -  
 + - 2 - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - 1 - -   
 + - - -                                                                   
  -                                                                 Eag1-  
 - - - - - - - - 1 - + - - - - - - - - - - - + - - - - - - 1 - - - - + -   
 - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + -   
 - -                                                                       
  -                                                                        
 Eco47I- - - 3 - - - 1 1 - - + - - - 1 - - - - - - - + - - - - - - - - -   
 - - + - - 1 - - - - 2 1 - - + 1 - - - - - - - - - - + - - - - - 1 - - 1   
 - - + - - -                                                               
  -                                                             Eco52I- -  
 - - - - - - - 1 - + - - - - - - - - - - - + - - - - - - 1 - - - - + - -   
 - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - -   
 -                                                                         
  -                                                                       E
 co81I- - - - - - - - - - 1 + 1 - - - - - - - - - - + - - - - - - - - - -  
 - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - -   
 - + - - -                                                                 
  -                                                               EcoN1-   
 - - - - - - - - - 1 + - - - - - 1 - - - - - + - - - - - - - - - - - + -   
 - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + -   
 - -                                                                       
  -                                                                        
 EcoO109I- - - 1 - - - - - - - + - - - 1 - - - - - - - + - - - - - - - -   
 - - - + - - - - - - - - 1 - - + - - - - - - - - - - - + - - - - - - - -   
 - - - + - - -                                                             
  1                                                           EcoR1- - -   
 - - - - - - - - + - - - - - 1 - - - - - + - - - - - - - - - - - + - - -   
 - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - -   
 -                                                                         
  EcoR1*- - - - - - - - - - - 1 1 - - - - 1 - - - 2 1 + 1 1 1 1 1 - - - -  
 - - + - - - - - - - - - - - + - - - - - - - - - - - + - - 1 - - - 1 - -   
 1 - + - - -                                                               
  -                                                             EcoR2- -   
 - 1 - - - 1 - 2 - + - - 2 3 - - 3 1 - - - + - - 3 - - - - - - - - + 1 -   
 - - - - - - 1 - - + 1 - - - - 1 - 2 - - - + - - - - - - - - - - - + - -   
 -                                                                         
  -                                                                       E
 coR5- 1 1 - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - -   
 - + - - - - - - 1 1 - - - + - - - - - - - - - - - + - - - - - - - - - -   
 - + - - -                                                                 
  -                                                                        
      EcoT22I- - - - - - - - - - - + - - - - - - - - - - - + - - 1 1 - -   
 - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - -   
 - - - - - + - - -                                                         
  -                                                       Fdi2- - - - - -  
 - - - - - + - - - - - - - - - - - + - - - - - - - - - 1 - + - - - - - -   
 - - - - - + - - - - - - - - - - - + - - - - - - 1 - - - - + - - - -       
       Fnu4H1- - - - - - - - - 1 - 3 - - 1 - - - - - 1 - - + - - - - 1 1   
 1 2 2 2 3 + - 1 1 2 - - - - - - - + 1 1 2 - 3 1 - 1 - 1 2 + 1 - - - 1 -   
 1 - 2 - 1 + 1 - -                                                         
  -                                                       FnuD2- - - - 2   
 1 - - - - - 2 - - 1 - - - - - - - - + - - - - - - - - 1 - - + 2 - - - -   
 - - - - 2 - + 2 1 - - - 1 - - - - - + 1 - - - 1 - - - - - 1 + - - 1 -     
     Fok1- - - - - 1 - - - - - + - - 1 - 1 - - 1 - - - + - - - 1 - - 1 -   
 - - - 1 1 - - - - - - - - - 1 + - 1 - - - - - - - - - + - - - 1 - 1 - -   
 - 1 - + - - -                                                             
  -                                                           Fsp1- - - -  
 - - - - - - - + - - - - - - - - - - - + - - - - - - - - - 1 - + - - - -   
 - - - - - - - + - - - - - - - - - - - + - - - - - - 1 - - - - + - - - -   
   Hae2- - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - 1 -   
 - - + - - - - - - - - - 1 - + - - - 1 - - - - 1 - - + - - - - - - - - -   
 - - + - - -                                                               
  -                                                             Hea3- - 3  
 - - - - 1 - 3 - + 1 1 2 - - - 1 1 - - 1 1 - - - - 2 2 1 - 1 - - + - 2 -   
 - - - 2 2 - - - + - - - - - 3 - - - - - 1 - - - - 1 1 - - 1 - - + - - -   
 1                                                                         
  Hap2- 1 1 - - 1 - 2 - 1 - + - - - - - - - - - - - + - - - - - - 1 1 2 1  
 1 1 - 1 - - - - 1 1 - - 1 1 1 2 - - - - - 1 - 2 - + 1 - - - 1 1 1 1 - -   
 1 + - - -                                                                 
  -                                                               Hga1- -  
 - - - 1 - - 1 - - 1 - - - - - - 1 - - - - + - - - - - - - - - - - + - -   
 - - - - - - - - - + - 1 - - - - 1 - - - - + - 1 - - - - - - - - 1 + - -   
 -                                                                         
  -                                                                       H
 giA1- - - - 1 - - - - - - + - - - - - - - - 1 - - + - - - - - - - - - 1   
 - 1 - - - - - - - - - 1 - + - - - 1 - - - - - 1 - + - - - - - - - - - -   
 1 1 - - -                                                                 
  -                                                               Hha1- -  
 - - 2 2 - - 1 - - 1 - - - - - - - - - - - + - - - - - - - 2 1 1 - + 3 -   
 - 1 - - - - - 3 - + 1 1 1 1 1 - - 1 1 1 - 1 1 - - - - 1 1 - - - 1 + - -   
 1 -                                                                       
  HinP1- - - - 2 2 - - 1 - - 1 - - - - - - - - - - - + - - - - - - - 2 1   
 1 - + 3 - - 1 - - - - - 3 - + 1 1 1 1 1 - - 1 1 1 - 1 1 - - - - 1 1 - -   
 - 1 + - -                                                                 
  1 -                                                             Hinc2-   
 - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + -   
 - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - 1 + -   
 - -                                                                       
  -                                                                        
 Hind2- - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - -  
 - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - -   
 1 + - - -                                                                 
  -                                                               Hind3-   
 - - - - - - - - - - + - - - - - - - - - - - + - - - - - 1 - - - - - + -   
 - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + -   
 - -                                                                       
  -                                                                        
 Hinf1- - - 1 - - 1 - - - 2 + - - - - - - 1 - - 1 - + - - - - - - - - - -  
 - + - 1 - 2 - - - - - - 1 + - - - - 1 1 - - - 1 - + - - - 1 - - - - - -   
 - + - - -                                                                 
  -                                                               Hpa2- 1  
 1 - - 1 - 2 - 1 - + - - - - - - - - - - - + - - - - - - 1 1 2 1 1 1 - 1   
 - - - - 1 1 - - 1 1 1 2 - - - - - 1 - 2 - + 1 - - - 1 1 1 1 - - 1 + - -   
 -                                                                         
  -                                                                       H
 ph1- - - - - - - - - - - + - - 2 - - - - - - 1 - + - - - - - - - - - - 1  
 + - - - - - - - - - - - + 2 - - - - - - - - - - + - - 1 - 1 - - - - 1 -   
 + 2 - -                                                                   
  -                                                                 Kpn1-  
 - - - - 1 - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + -   
 - - - - - - - - - 1 + - - - - - - - - - - - + - - - - - - - - - - - + -   
 - -                                                                       
  -                                                                        
 Mae11 - - 1 - - - - 3 - - + - - - - - 1 - 1 - - 1 1 - - - - - 1 - - - -   
 - + - - - - 1 - - - 1 - - + - - - - - - - - - - - 1 - - 1 - - - 1 - - -   
 - + - - -                                                                 
  -                                                               Mae2- -  
 - - - - - - - 1 - + - 1 1 - - - - - - - - 1 - - - - - - - - - 1 - 1 - -   
 - - - - - - - - - + - - 1 - - - - - - - - + - 1 - - - - 1 - - - - 1 - -   
 1 -                                                                       
  Mae31 - - - - - - - - - - + 2 1 - 1 - 1 - - 1 - - + - - - - - - - - - 1  
 - + - 1 - - - 1 - - - - - + - 1 1 - - - - - - 1 1 1 - - - 1 - - - 2 - 1   
 - 1 - - -                                                                 
  -                                                               Mbo1- 1  
 - - - - - - - - - + - 1 1 - 1 - - 1 - - - 1 - 1 - - - - 3 - - 1 1 2 1 -   
 - - - 1 - - - - - 1 - - - - - - - - - - - + 2 2 2 1 - - - 2 1 - - 2 1 -   
 -                                                                         
  -                                                                       M
 ae11 - - - - - - - - - - + 1 - 3 - - - - - - 2 - 2 - 2 - - - - - - - - -  
 + - - - - - 1 - - - - - + - - 1 - - - - - - - - + - 1 3 1 - - 2 - - - 1   
 + - - -                                                                   
  1                                                                 Msp1- 1
  1 - - 1 - 2 - 1 - + - - - - - - - - - - - + - - - - - - 1 1 2 1 1 1 - 1  
 - - - - 1 1 - - 1 1 1 2 - - - - - 1 - 2 - + 1 - - - 1 1 1 1 - - 1 + - -   
 -                                                                         
  -                                                                       M
 st1- - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - 1 -  
 + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - 1 - - - -   
 + - - -                                                                   
  -                                                                 Mst2-  
 - - - - - - - - - 1 + 1 - - - - - - - - - - + - - - - - - - - - - - + -   
 - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + -   
 - -                                                                       
  -                                                                        
 Mva1- - - 1 - - - 1 - 2 - + - - 2 3 - - 3 1 - - - + - - 3 - - - - - - -   
 - + 1 - - - - - - - 1 - - + 1 - - - - 1 - 2 - - - + - - - - - - - - - -   
 - + - - -                                                                 
  -                                                               Nae1- -  
 - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - 1   
 - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - -   
 -                                                                         
  -                                                                       N
 ar1- - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - 1 - - -  
 + - - - - - - - - - 1 - + - - - - - - - - - - - + - - - - - - - - - - -   
 + - - -                                                                   
  -                                                                 Nci1- 1
  1 - - 2 - 2 - 1 - + - - - - - - - - - - - + - - - - - - - - 1 1 - + - -  
 - - - - 1 1 - - 2 1 1 2 - - - - - - - 1 - + - - - - - - 1 - - - 1 + - -   
 -                                                                         
  -                                                                       N
 co1- - - - - - - - - - - + - - - - - - - - - - - + - - - - 1 - - - - - -  
 + - 1 - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - -   
 + - - -                                                                   
  -                                                                 Nde1-  
 - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + -   
 - - - - - - - - - - + - - - 1 - - - - - - - + - - - - - - - - - - - + -   
 - -                                                                       
  -                                                                        
 Nde2- 1 - - - - - - - - - + - 1 1 - 1 - - 1 - - - 1 - 1 - - - - 3 - - 1   
 1 2 1 - - - - 1 - - - - - 1 - - - - - - - - - - - + 2 2 2 1 - - - 2 1 -   
 - 2 1 - -                                                                 
  -                                                               Nhe11 -  
 - - - - - - - - - + - - - - - - - 1 - - - + - - - - - - - - - - - + - -   
 - - 1 - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - -   
 -                                                                         
  -                                                                       N
 la31 - - - - - - - - - - + - - - 1 - - 1 1 - 1 1 + 1 - 1 1 1 - 1 - - - 1  
 + 1 2 - - - 1 - - - - - + - 1 1 - - 1 - - - - - + - 1 - - - - - 2 1 1 -   
 + - 1 -                                                                   
  1                                                                 Nla4- 1
  1 2 - 1 - 1 - 2 - 1 - - 1 1 - - - - - - - + - 1 1 1 - - - 1 1 - - + - -  
 - - - - - 1 1 1 1 + 1 - - - - 1 1 - - - - + - - - 1 2 - - 1 - - - + - -   
 1 -                                                                       
  Nsi1- - - - - - - - - - - + - - - - - - - - - - - + - - 1 1 - - - - - -  
 - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - -   
 - + - - -                                                                 
  -                                                                        
 Nsp(7524)1- - - - - - - - - - - + - - - - - - 1 - - 1 1 + - - 1 1 - - -   
 - - - - + 1 - - - - - - - - - - + - 1 - - - 1 - - - - - + - - - - - - -   
 - - - - + - - -                                                           
  -                                                         Nsp(7524)2- -  
 - 1 1 1 - 1 - - - + - - - - - - 1 - 1 - - + - - - - - - - 1 1 1 - 1 1 -   
 - - - - - - 1 2 - 1 - - - 1 - - - - - 1 - + - - - - - - - - - - 1 1 - -   
 -                                                                         
  -                                                                       N
 spB2- 1 1 - - - - - - - - + - - - - - - - - 1 - - + - 1 - - - - - - - 1   
 - + - - - - - - 2 - - - - 1 - 1 - - - - - - - 1 - + 1 - - - - - - - - -   
 - 1 - - -                                                                 
  -                                                               NspH1-   
 - - - - - - - - - - + - - - - - - 1 - - 1 1 + - - 1 1 - - - - - - - + 1   
 - - - - - - - - - - + - 1 - - - 1 - - - - - + - - - - - - - - - - - + -   
 - -                                                                       
  -                                                                        
 PaeR7I- - - - - - - - - - - + - - - - - - - - - - - 1 - - - - - - - - -   
 - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - -   
 - - + - - -                                                               
  -                                                             Pal1- - 3  
 - - - - 1 - 3 - + 1 1 2 - - - 1 1 - - 1 1 - - - - 2 2 1 - 1 - - + - 2 -   
 - - - 2 2 - - - + - - - - - 3 - - - - - 1 - - - - 1 1 - - 1 - - + - - -   
 1                                                                         
  Ple1- - - - - - - - - - 1 + - - - - - - 1 - - - - + - - - - - - - - - -  
 - + - - - 1 - - - - - - 1 + - - - - 1 - - - - 1 - + - - - 1 - - - - - -   
 - + - - -                                                                 
  -                                                                        
      PpuM1- - - 1 - - - - - - - + - - - 1 - - - - - - - + - - - - - - -   
 - - - - + - - - - - - - - 1 - - + - - - - - - - - - - - + - - - - - - -   
 - - - - + - - -                                                           
  -                                                         Pss1- - - 1 -  
 - - - - - - + - - - 1 - - - - - - - + - - - - - - - - - - - + - - - - -   
 - - - 1 - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - 1     
     Pst1- - - - - - - - - - - 1 - 1 - - - - - - - - 1 + - - - - - 1 - -   
 1 - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - 1 -   
 - - - + - - -                                                             
  -                                                           Pvu1- - - -  
 - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - -   
 - - - - - - - + - - - - - - - - - - - + - - - - - - - - 1 - - + - - - -   
   Pvu2- 1 1 - - - - - - - - + - - - - - - - - 1 - - + - - - - - - - - -   
 1 - + - - - - - - 2 - - - - + - - - - - - - - - - - + - - - - - - - - -   
 - - + - - -                                                               
  -                                                             Rsa1- - -  
 - 1 1 - - 1 - - + - - - 1 - - 1 1 - - - + - - - - - - - - - - - 1 - - -   
 - - - - - - - 1 + - - - 1 - - - - - - - + - - - - - - - - - 1 - + - - -   
 -                                                                         
  Rsr2- - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - -  
 - + - - 1 - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - -   
 - + - - -                                                                 
  -                                                               Sac1- -  
 - - 1 - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - -   
 - - - - - - - 1 - + - - - - - - - - - - - + - - - - - - - - - - - + - -   
 -                                                                         
  -                                                                       S
 au1- - - - - - - - - - 1 + 1 - - - - - - - - - - + - - - - - - - - - - -  
 + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - -   
 + - - -                                                                   
  -                                                                        
 Sau3A1- 1 - - - - - - - - - + - 1 1 - 1 - - 1 - - - 1 - 1 - - - - 3 - -   
 1 1 2 1 - - - - 1 - - - - - 1 - - - - - - - - - - - + 2 2 2 1 - - - 2 1   
 - - 2 1 - -                                                               
  -                                                             Sau96I- - 3
  3 - - - 1 1 2 - + 1 - 1 1 - - 1 1 - - - + - - - - - - - - - - - + - - 1  
 - - - 2 4 1 - - + 1 - - - - - - - - - - + - - - - 1 2 - - 1 - - + - - -   
 1                                                                         
  Sca1- - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - -  
 - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - 1   
 - + - - -                                                                 
  -                                                               ScrF1-   
 1 1 1 - 2 - 3 - 3 - + - - 2 3 - - 3 1 - - - + - - 3 - - - - - 1 1 - + 1   
 - - - - - 1 1 1 - 2 1 2 2 - - - 1 - 2 - 1 - + - - - - - - 1 - - - 1 + -   
 - -                                                                       
  -                                                                        
 Sdu1- - - 1 1 1 - 1 - - - + - - - - - - 1 - 1 - - + - - - - - - - 1 1 1   
 - 1 1 - - - - - - - 1 2 - 1 - - - 1 - - - - - 1 - + - - - - - - - - - -   
 1 1 - - -                                                                 
  -                                                               Sec1- 1  
 1 2 - 1 1 2 - 5 - + - - 1 4 - - 2 - - - - 1 - - 2 - 3 1 - - - 1 - + - 1   
 - - - - 1 1 2 - 1 1 2 - - - - - - 1 - - - + - - - - - - - - - - - + - -   
 -                                                                         
  -                                                                       S
 faN1- - - 1 - - 1 - - - - 1 - - - - - - - 1 - - - + - 1 1 1 - - - 1 - -   
 1 1 1 1 - 1 - - - 1 - - 1 + - 1 1 2 - - 1 - - - - + - - - - - - - 1 - 1   
 - + 1 - -                                                                 
  -                                                               Sfi1- -  
 - - - - - - - - - + - - - - - - - - - - - + - - - - 1 - - - - - - + - -   
 - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - -   
 -                                                                         
  -                                                                       S
 ma1- - - - - 1 - - - - - + - - - - - - - - - - - + - - - - - - - - - - -  
 + - - - - - - - - - - 1 + - - - - - - - - - - - + - - - - - - - - - - -   
 + - - -                                                                   
  -                                                                 Spe1-  
 - - - - - - - 1 - - + - - - - - - - - - - - + - - - - - - - - - - - + -   
 - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + -   
 - -                                                                       
  -                                                                        
 Sph1- - - - - - - - - - - + - - - - - - - - - 1 - + - - 1 1 - - - - - -   
 - + 1 - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - -   
 - + - - -                                                                 
  -                                                               Ssp1- -  
 - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - -   
 - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - 1   
 -                                                                         
  -                                                                       S
 st1- - - - 1 - - - - - - + - - - - - - - - - - - + - - - - - - - - - - -  
 + - - - - - - - - - 1 - + - - - - - - - - - - - + - - - - - - - - - - -   
 + - - -                                                                   
  -                                                                 Stu1-  
 - - - - - - - - - - + - - - - - - - - - - - 1 - - - - - 1 - - - - - + -   
 - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + -   
 - -                                                                       
  -                                                                        
 Sty1- - - 1 - - 1 - - - - + - - - 1 - - - - - - - 1 - - - - 1 1 - - - -   
 - + - 1 - - - - - - 1 - - 1 - - - - - - - - - - - + - - - - - - - - - -   
 - + - - -                                                                 
  -                                                               Tag1- -  
 - - - - - - - - 1 + - 1 - - 2 - - 1 - - - 1 - - - - - 1 - - - 1 - 3 - 1   
 - - 1 - - - - - - + - - - - - - 1 - - - - + - - - - - - - - - - - 1 - -   
 -                                                                         
  -                                                                       T
 ha1- - - - 2 1 - - - - - 2 - - 1 - - - - - - - - + - - - - - - - - 1 - -  
 + 2 - - - - - - - - 2 - + 2 1 - - - 1 - - - - - + 1 - - - 1 - - - - - 1   
 + - -                                                                     
  1 -                                                                      
 Tthl11I- - - - - 1 - - - - 1 + - - - 1 - - - - - - - + - - - - - - - - -  
 1 - + - - - - - - - - - - 1 + - - 1 - - - - - - - - + - - - - - - - - -   
 - - + - - -                                                               
  -                                                             Xba1- - -  
 - - - - - - - - + - - - - - - - - - - 1 + - - - - - - - - - - - + - - -   
 - - - - - 1 - - + - - - - - - - - - - - + - - - - - - - - - - - + - - -   
 -                                                                         
  Xho1- - - - - - - - - - - + - - - - - - - - - - - 1 - - - - - - - - - -  
 - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - -   
 - + - - -                                                                 
  -                                                               Xho2- -  
 - - - - - - - - - + - - - - - - - - - - - 1 - 1 - - - - 1 - - 1 - + 1 -   
 - - - - - - - - - + - - - - - - - - - - - + 1 1 2 - - - - - - - - 2 - -   
 -                                                                         
  -                                                                       X
 ma1- - - - - 1 - - - - - + - - - - - - - - - - - + - - - - - - - - - - -  
 + - - - - - - - - - - 1 + - - - - - - - - - - - + - - - - - - - - - - -   
 + - - -                                                                   
  -                                                                 Xma3-  
 - - - - - - - - 1 - + - - - - - - - - - - - + - - - - - - 1 - - - - + -   
 - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + -   
 - -                                                                       
  -                                                                        
 Xmn1- - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - -   
 - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - -   
 - 1 - - -                                                                 
  -                                                               Xor2- -  
 - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - -   
 - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - 1 - - + - -   
 -                                                                         
  -                                                                        

                                  TABLE 6                                 
__________________________________________________________________________
from 1 to 6365. Numbered from position 1.                                 
 ##STR2##                                                                 
 ##STR3##                                                                 
 ##STR4##                                                                 
 ##STR5##                                                                 
 ##STR6##                                                                 
 ##STR7##                                                                 
 ##STR8##                                                                 
 ##STR9##                                                                 
 ##STR10##                                                                
 ##STR11##                                                                
 ##STR12##                                                                
 ##STR13##                                                                
 ##STR14##                                                                
 ##STR15##                                                                
 ##STR16##                                                                
 ##STR17##                                                                
 ##STR18##                                                                
 ##STR19##                                                                
 ##STR20##                                                                
 ##STR21##                                                                
 ##STR22##                                                                
 ##STR23##                                                                
 ##STR24##                                                                
 ##STR25##                                                                
 ##STR26##                                                                
 ##STR27##                                                                
 ##STR28##                                                                
 ##STR29##                                                                
 ##STR30##                                                                
 ##STR31##                                                                
 ##STR32##                                                                
 ##STR33##                                                                
 ##STR34##                                                                
 ##STR35##                                                                
 ##STR36##                                                                
 ##STR37##                                                                
 ##STR38##                                                                
 ##STR39##                                                                
 ##STR40##                                                                
 ##STR41##                                                                
 ##STR42##                                                                
 ##STR43##                                                                
 ##STR44##                                                                
 ##STR45##                                                                
 ##STR46##                                                                
 ##STR47##                                                                
 ##STR48##                                                                
 ##STR49##                                                                
 ##STR50##                                                                
 ##STR51##                                                                
 ##STR52##                                                                
 ##STR53##                                                                
 ##STR54##                                                                
 ##STR55##                                                                
 ##STR56##                                                                
 ##STR57##                                                                
 ##STR58##                                                                
 ##STR59##                                                                
 ##STR60##                                                                
 ##STR61##                                                                
 ##STR62##                                                                
 ##STR63##                                                                
 ##STR64##                                                                
 ##STR65##                                                                
 ##STR66##                                                                
 ##STR67##                                                                
 ##STR68##                                                                
 ##STR69##                                                                
 ##STR70##                                                                
 ##STR71##                                                                
 ##STR72##                                                                
 ##STR73##                                                                
 ##STR74##                                                                
 ##STR75##                                                                
 ##STR76##                                                                
 ##STR77##                                                                
 ##STR78##                                                                
 ##STR79##                                                                
 ##STR80##                                                                
 ##STR81##                                                                
 ##STR82##                                                                
 ##STR83##                                                                
 ##STR84##                                                                
 ##STR85##                                                                
 ##STR86##                                                                
 ##STR87##                                                                
 ##STR88##                                                                
 ##STR89##                                                                
 ##STR90##                                                                
 ##STR91##                                                                
 ##STR92##                                                                
 ##STR93##                                                                
 ##STR94##                                                                
__________________________________________________________________________
 zymes which do not cut LXSNRIIL2:                                        
  -                                                                       
 ##STR95##
To generate the LXSN-RI-IL2 retroviral vector, 10 micrograms of pLXSN-RI-IL2 DNA was transfected into the ecotropic packaging cell line PE501 by standard calcium phosphate precipitation methods (Miller et al., Mol. Cell Biol. 6:2895, 1986). The transfected PE501 cell line was grown in DMEM medium with 10% FCS. The medium was changed after 24 hours and supernatant harvested 24 hours later to infect the amphotropic packaging cell line PA317 as described (Miller et al., Mol. Cell Biol. 6:2895, 1986 and Miller et al., BioTechniques 7:980, 1989). The infected PA317 cells were harvested by trypsinization 24 hours later and replated 1:20 in DMEM containing 10% FCS and the neomycin analogue G418 (400 μg/ml). The cells were grown at 37° C. in 7% CO2 atmosphere. The selection medium was changed every 5 days until colonies appeared. On day 14, twenty colonies were selected, expanded and tested for vital production by standard methods (Xu et al., Virology 171:331-341, 1989). Briefly, supernatants were harvested from confluent culture dishes, passed through a 0.45 μm filter, diluted with DMEM with 10% FCS and utilized to infect NIH 3T3 cells in the presence of 8 μg/ml polybrene. After 24 hours, the infected NIH 3T3 cells were grown in culture medium that contained the neomycin analogue G418. After 12-14 days, the colonies were stained, counted and the viral titer calculated as described (Xu et al., Virology 171:331-341, 1989).
Colonies with the highest viral titers (>104 infectious units/ml) were tested for IL-2 expression by Northern blot analyses. Colonies with the highest viral titers and documented IL-2 expression were cryopreserved and will be utilized as stock cultures to produce the LXSN-RI-IL2 retroviral vector trial.
EXAMPLE IV Retroviral Vector Construction and Cytokine Expression
To increase IL-2 production by transduced cell lines, vectors were used containing different promoters to drive IL-2 expression, and a human IL-2 cDNA was directionally sub-cloned into the insulin secretory signal peptide (17). The IL-2 cDNA was directionally sub-cloned into the parental plasmids of the LXSN (LTR promoter) and LNCX (CMV promoter) vectors (gifts of Dr. A. D. Miller) (18). The newly constructed vectors (FIG. 1), designated as LXSN-IL2 and LNCX-IL2, were packaged in the PA317 cell line for production of retroviral supernatant. As a control, the high level expressing, double copy vector DC/TKIL-2 vector (thymidine kinase promoter) (a gift of Dr. E. Gilboa) was used for comparison.
These vectors were used to transduce a number of murine and human, primary and established cell lines. Pools of transduced cells were selected and expanded in DMEM medium, containing 10% fetal bovine serum (FBS) and 400 μg/ml of active G-418, a neomycin analogue. The results of expression studies in the MCR9 and Balb/c 3T3 cell lines are presented in Table 7.
              TABLE 7                                                     
______________________________________                                    
Comparison of IL-2 expression by fibroblasts                              
transduced with different IL-2 vectors.                                   
                 ng IL-2  Units IL-2                                      
Fibroblast                                                                
          Vector       per 10.sup.6 cells per day                         
______________________________________                                    
Murine    LNCX (Control)                                                  
                        0.4 ± 50%                                      
                                  <1                                      
          LNCX-IL2     33.7 ± 11%                                      
                                  67                                      
          LXSN-IL2     6.6 ± 6%                                        
                                  13                                      
          DC/TKIL-2    1.9 ± 5%                                        
                                  4                                       
Human     LXSN (Control)                                                  
                        0.7 ± 29%                                      
                                  1                                       
          LNCX-IL-2    159.5 ± 17%                                     
                                  319                                     
          LXSN-IL2     25.5 ± 15%                                      
                                  51                                      
          DC/TKIL-2     3.0 ± 10%                                      
                                  6                                       
______________________________________
EXAMPLE V Fibroblast Culture and Conditions for Retroviral Transduction
The culture conditions for the growth of primary fibroblasts retroviral transduction were optimized. Primary fibroblasts were successfully cultured. The optimal conditions enable the growth of approximately 3-4×106 primary fibroblasts from a 12 mm2 skin biopsy in approximately 4-6 weeks. Retroviral infection, G418 selection, and expansion of the genetically modified fibroblasts takes an additional 4-6 weeks.
Exploring the conditions for genetic modification of primary fibroblasts suggests that optimal transduction may be obtained by the following procedure: The fibroblasts are synchronized in G1 phase by serum starvation, followed by stimulation with medium containing 15% fetal bovine serum 15 hours prior to transduction. The cells are then subjected to 2 cycles of retrovirus infection, each cycle lasting approximately 3 hours. The cells are refed with fresh media overnight, and then selection in G418 is initiated the next day. This method is capable of transducing 5-15% of the fibroblasts in a culture, depending on the multiplicity of infection.
This procedure was used to transduce a large number of primary and established fibroblasts. As an example, Table 8 compares the expression levels of IL-2 in fibroblast lines transduced with LXSN-IL2.
              TABLE 8                                                     
______________________________________                                    
Expression of IL-2 by fibroblasts transduced with LXSN-IL2.               
Fibroblast              ng IL-2   Units IL-2                              
Line      Species Origin    per 10.sup.6 cells per day                    
______________________________________                                    
Balb/c 3T3                                                                
          Murine  Transformed                                             
                            6.6 ± 6%                                   
                                    13                                    
MCR9      Human   Embryonic 25.5 ± 15%                                 
                                    51                                    
NHDF 313  Human   Skin      25.0 ± 10%                                 
                                    50                                    
GT1       Human   Skin      15.0 ± 5%                                  
                                    30                                    
______________________________________
These results indicate that the IL-2 expression levels in established, embryonic, and primary fibroblast cultures are similar. Comparison of these data with Table 7 suggest that IL-2 expression is affected more by factors such as different promoters than by the fibroblast line used. Similarly, changes in culture conditions can have important effects on IL-2 expression. Table 9 shows that transduced GT1 cells, a primary human fibroblast culture expressed 15-fold more IL-2 under 100 μg/ml G418 selection than under 25 μg/ml G418 selection. Several other primary fibroblast lines have also been transduced with our vectors and are currently growing under G418 selection.
              TABLE 9                                                     
______________________________________                                    
Effect of G418 concentration on IL-2 expression by GT1                    
cells transduced with LXSN-IL2.                                           
Selection dose                                                            
              ng IL-2 secreted                                            
of G418       per 10.sup.6 cells per day*                                 
______________________________________                                    
25 μg/ml   1.0 ± 10%                                                
50 μg/ml   3.0 ± 6%                                                 
100 μg/ml  15.0 ± 5%                                                
______________________________________                                    
 *After three weeks of G418 selection.
EXAMPLE VI Comparison of IL-2 Expression Levels Induced Peripheral Blood Lymphocytes and Genetically Modified Fibroblasts
In order to compare the production of IL-2 by genetically modified fibroblasts to that achieved by stimulating normal human peripheral blood lymphocytes (nPBL) in vitro, nPBL were isolated by Ficol-Paque density centrifugation, and cultured in the presence of allogeneic nPBL (mixed lymphocyte culture, MLC) or 2 μM calcium ionophore (CI) (A23187) free acid) plus 17 nM phorbol 12-myristate 13-acetate (PMA). The results of this experiment, present in Table 10, indicate that the level of IL-2 expression in the PMA/CI stimulated normal T cell population was 2 ng/106 cells/24 hours. This is equivalent to IL-2 expression by Balb/c 3T3 fibroblasts transduced with DC/TKIL-2 (Table 7), our least productive vector. The level of IL-2 expression in the MLC was 130 pg/106 cells/24 hours. This was lower than the PMA/CI stimulated culture, presumably because PMA/CI induced a nonspecific response while MLC resulted in specific Th stimulation. When the estimated percentage of antigen-specific Th in the MLC-stimulated population is taken into consideration, the level of IL-2 expression per stimulated T cell becomes equivalent for both methods.
              TABLE 10                                                    
______________________________________                                    
Levels of IL-2 secretion by different cells.                              
                  pg IL-2 secreted                                        
Cells             per 10.sup.6 cells per day                              
______________________________________                                    
Lymphocytes:                                                              
Control (non-activated)                                                   
                        5 ± 50%                                        
PMA + Calcium Ionophore                                                   
                   2,000 ± 6%                                          
Mixed lymphocyte culture                                                  
                      130 ± 90%                                        
Transduced fibroblasts:                                                   
MCR9-LXSN-IL2     24,000 ± 5%                                          
MCR9-LNCX-IL2     162,000 ± 20%                                        
MCR9-DC/TKIL-2    10,000 ± 6%                                          
______________________________________
EXAMPLE VII
Fibroblast Mediated Cytokine Gene Therapy in Murine Tumor Models
Two experimental protocols were used to study the efficacy of fibroblast-mediated cytokine gene therapy on induction of anti-tumor immunity. The first protocol was designed to test the effects of genetically modified fibroblasts on tumor implantation, while the second protocol was designed to induce a systemic anti-tumor immunity. The results of each experiment are presented with two figures and one table. In the first figure, the rate of tumor growth for each treatment group is presented as the mean tumor size in the group over time. In the second figure, a Kaplan-Meier curve presents the time of tumor onset for the individual animals in each treatment group. The number of animals, the number and percentage of tumor free animals, and the tumor size distribution patterns for each experiment are presented in a table.
EXAMPLE VII Effect of Fibroblast Mediated Cytokine Gene Therapy on Tumor Implantation
Mice were injected subcutaneously with mixtures of 5×104 CT26 cells and 2×106 fibroblasts genetically modified by different retroviral vectors to express IL-2. In the control arms injected with tumor cells only, or with tumor cells mixed with unmodified fibroblasts, 31 of 33 animals (94%) developed tumors by 4 weeks (FIGS. 6 and 7, Table 9). In contrast, 22 out of the 34 animals (65%) receiving fibroblast mediated cytokine gene therapy were tumor free at 3 weeks, and 5 animals (18%) remain tumor free after 12 weeks. Those animals that received fibroblast mediated IL-2 therapy and developed tumor were characterized by a delayed onset and rate of tumor growth.
                                  TABLE 11                                
__________________________________________________________________________
Effect of IL-2 modified fibroblasts on tumor establishment and            
development.                                                              
2 × 10.sup.6 fibroblasts mixed with 5 × 10.sup.4 CT26 tumor   
cells at time of injection.                                               
           Animal Number                                                  
Fibroblasts   Tumor-                                                      
                  Tumor-                                                  
                      Percent                                             
                            Tumor Size (mm.sup.2)                         
                                             Median Tumor Size            
mixed with tumor cells                                                    
           Total                                                          
              free                                                        
                  bearing                                                 
                      Tumor-free                                          
                            25-100                                        
                                101-200                                   
                                     201-300                              
                                          >301                            
                                             (mm.sup.2)                   
__________________________________________________________________________
After 12 Weeks:*                                                          
Control (no fibroblasts)                                                  
           11 0   11   0%   1   0    1    9  420 ± 145                 
Unmodified fibroblasts**                                                  
           13 2   11  15%   1   0    1    7  389 ± 265                 
DCTK-IL2 fibroblasts                                                      
           13 0   13   0%   1   3    5    4  267 ± 168                 
LNCX-IL2 fibroblasts                                                      
           13 5    8  39%   5   2    0    1  72 ± 90                   
__________________________________________________________________________
 * Mean tumor size is for 4 weeks, the last timepoint at which tumors were
 measured.                                                                
 **Two mice in this arm developed intraperitoneal tumors which were not   
 measurable.
After 3 weeks the mean tumor size (measured as the product of the longest and widest tumor axes) in the control group of mice was 128 mm2, compared to 68 and 7 mm2 in groups of mice injected with tumor cells mixed with fibroblasts transduced with DC/TKIL-2 or LNCX-IL2, respectively. This resulted in a highly significant difference (corrected x2 =18.69, p=0.001) between the IL-2 treated animals compared to the mice treated with CT26 alone or CT26 mixed with unmodified fibroblasts. After four weeks the equivalent measurements were 373,300 and 72 mm2 (Table 11). It is notable that LNCX-IL2, the highest expressing vector caused substantially greater inhibition of tumorigenicity than the lower expressing vector DC/TKIL-2. A multivariate non-parametric statistical procedure (19,20), utilized to evaluate differences in tumor growth, demonstrated that after 4 weeks the differences between the growth curves for the four groups presented in FIG. 2 were highly significant (p<0.001). Subsequent comparisons between the control arm and animals that received tumor cells mixed with IL-2 transduced fibroblasts revealed a significant difference (P<0.05). The differences between the animals injected with tumor cells alone, and those injected with tumor cells plus unmodified fibroblasts were not significant, while the differences between animals receiving low IL-2 expressing fibroblast, and those receiving high IL-2 expressing fibroblasts was significant (P=0.05).
When mice were injected with 2×106 modified fibroblasts mixed with 1×105 live tumor cells the results became more striking (see FIGS. 8 and 9, and Table 12). All the control animals developed tumors after 4 weeks whereas 33% and 27% of the animals treated with fibroblasts modified with the DCTK-IL2 or LXSN-IL2 vectors (respectively) remain tumor free after 7 weeks (the experiment is ongoing). More dramatically, 75% of the animals treated with fibroblasts modified with the highest IL-2 producing vector, LNCX-IL2, remain tumor free after 7 weeks. These data clearly demonstrate the importance of an initial high dose of IL-2 to prevent tumor establishment.
                                  TABLE 12                                
__________________________________________________________________________
Effect of IL-2 modified fibroblasts on tumor establishment and            
development.                                                              
2 × 10.sup.6 fibroblasts mixed with 1 × 10.sup.5 CT26 tumor   
cells at time of injection.                                               
            Animal Number                                                 
Fibroblasts    Tumor-                                                     
                   Tumor-                                                 
                       Percent                                            
                             Tumor Size (mm.sup.2)                        
                                              Mean Tumor Size             
mixed with tumor cells                                                    
            Total                                                         
               free                                                       
                   bearing                                                
                       Tumor-free                                         
                             25-100                                       
                                 101-200                                  
                                      201-300                             
                                           >301                           
                                              (mm.sup.2)                  
__________________________________________________________________________
After 6 Weeks:*                                                           
Control (no fibroblasts)**                                                
            13 0   13   0%   0   5    2    5  315 ± 197                
Unmodified fibroblasts**                                                  
            20 0   20   0%   0   2    3    14 350 ± 100                
DCTK-IL2 fibroblasts                                                      
            12 4    8  33%   0   1    4    3  185 ± 141                
LXSN-IL2 fibroblasts***                                                   
            15 4   11  27%   0   5    1    2  135 ± 121                
LNCX-IL2 fibroblasts                                                      
             8 6    2  75%   2   0    0    0   8 ± 14                  
__________________________________________________________________________
 * Mean tumor size is for 4 weeks, the last timepoint at which tumors were
 measured.                                                                
 **One mouse in each of these arms developed an intraperitoneal tumor whic
 was not measurable.                                                      
 ***Three mice in this arm developed intraperitoneal tumors which were not
 measurable.
As an additional control, mice were injected with CT26 cells genetically modified to express IL-2 (results not shown). Injection of up to 1×106 IL-2 expressing tumor cells into Balb/c mice failed to produce tumors. Injection of higher numbers however, resulted in some animals developing tumors with delayed onset. These data confirm the results reported in the literature (1). In order to compare the efficacy of IL-2 producing fibroblasts to IL-2 producing tumor cells, we mixed 2×106 CT26 tumor cells modified with the DCTK-IL2 vector with 1×105 unmodified tumor cells. FIGS. 10 and 11, and Table 13 show that DCTK-IL2 modified tumor cells are somewhat effective in preventing tumor development. Four weeks after injection, the mean tumor size for the treatment arm is 303 mm2, compared to 620 mm2 for the control arm. After 22 weeks, one animal (10%) remains tumor free, compared to none in the control arms. Data for animals treated under the same conditions with DCTK-IL2 modified fibroblasts in a separate experiment are included for comparison purposes. This comparison suggests that DCTK-IL2 modified tumor cells have an effect on tumor establishment similar to that of DCTK-IL2 modified fibroblasts.
                                  TABLE 13                                
__________________________________________________________________________
Effect of IL-2 modified cells on tumor establishment and developement.    
2 × 10.sup.6 DCTK-IL2-modified CT26 tumor cells mixed with 1        
× 10.sup.5 CT26 cells compared to 2 × 10.sup.6                
DCTK-IL2-modified fibroblasts mixed with 1 × 10.sup.5 CT26.         
            Animal Number                                                 
Cells          Tumor-                                                     
                   Tumor-                                                 
                       Percent                                            
                             Tumor Size (mm.sup.2)                        
                                              Mean Tumor Size             
mixed with tumor cells                                                    
            Total                                                         
               free                                                       
                   bearing                                                
                       Tumor-free                                         
                             25-100                                       
                                 101-200                                  
                                      201-300                             
                                           >301                           
                                              (mm.sup.2)                  
__________________________________________________________________________
After 22 Weeks:*                                                          
Control (no fibroblasts)                                                  
            5  0   5    0%   0   0    0    5  620 ± 190                
Unmodified fibroblasts                                                    
            5  0   5    0%   0   0    0    5  587 ± 69                 
DCTK-IL2-modified                                                         
            10 1   9   10%   1   0    2    5  303 ± 179                
CT26 cells                                                                
DCTK-IL2-modified                                                         
            8  2   6   25%   0   1    2    3  214 ± 158                
fibroblasts                                                               
__________________________________________________________________________
 *Mean tumor size is for 4 weeks, the last timepoint at which tumors were 
 measured.
EXAMPLE VII(b) Effect of Fibroblast Mediate Cytokine Gene Therapy on Systemic Anti-tumor Immunity
Groups of Balb/c mice were immunized with 2.5×105 irradiated tumor cells either alone or mixed with 2×106 transduced or unmodified fibroblasts, and challenged one week later with 5×104 live tumor cells in the opposite flank. These results (FIGS. 12 and 13, and Table 14) demonstrate that immunization with irradiated tumor cells and transduced fibroblasts protect some animals against a live tumor challenge, but that the protection is only slightly better than that achieved by immunization with irradiated tumor cells alone or irradiated tumor cells mixed with unmodified fibroblasts.
                                  TABLE 14                                
__________________________________________________________________________
Effect of Il-2 modified fibroblasts on induction of sytemic anti-tumor    
immunity.                                                                 
Mice immunized with 2 × 10.sup.6 fibroblasts mixed with 2.5 × 
10.sup.5                                                                  
irradiated CT26 tumor cells 7 days prior to challenge with 5 ×      
10.sup.4 fresh tumor cells.                                               
            Animal Number                                                 
Fibroblasts mixed with                                                    
               Tumor-                                                     
                   Tumor-                                                 
                       Percent                                            
                             Tumor Size (mm.sup.2)                        
                                              Mean Tumor Size             
irradiated tumor cells                                                    
            Total                                                         
               free                                                       
                   bearing                                                
                       Tumor-free                                         
                             25-100                                       
                                 101-200                                  
                                      201-300                             
                                           >301                           
                                              (mm.sup.2)                  
__________________________________________________________________________
After 22 Weeks:*                                                          
Control (saline)                                                          
            20 0   20   0%   0   0    1    19 574 ± 160                
Irradiated CT26 only**                                                    
            16 5   11  31%   2   1    2    5  250 ± 277                
Irradiated CT26 mixed with                                                
            15 4   11  27%   0   1    3    7  266 ± 199                
unmodified fibroblasts                                                    
DCTK-IL2 fibroblasts**                                                    
            25 10  15  40%   4   1    1    8  172 ± 194                
__________________________________________________________________________
 *Mean tumor size is for 4 weeks, the last timepoint at which tumors were 
 measured.                                                                
 ** One mouse in each of these arms developed an intraperitoneal tumor    
 which was not measurable.
In a second protocol similar to the one described above, animals were challenged with fresh tumor cells two weeks following immunization with irradiated tumor cells mixed with fibroblasts. The results, shown in FIGS. 14 and 15, and in Table 15, demonstrate that DCTK-IL2 modified fibroblasts mixed with irradiated tumor cells confers superior protection to subsequent tumor challenge than irradiated tumor cells alone, irradiated tumor cells mixed with unmodified fibroblasts, or irradiated tumor cells mixed with LNCX-modified fibroblasts. After 7 weeks, seven of ten animals (70%) treated with DCTK-IL2 modified fibroblasts remain tumor free compared to only one third of the control animals. At four weeks, the mean tumor size of this group was 41 mm2, compared to 180, 170, and 140 mm2 for the three control groups. Animals treated with LNCX-IL2 modified fibroblasts were also protected against subsequent tumor challenge, but the results were less striking. In this group, 54% of the animals remain tumor free and the mean tumor size for the group at four weeks was 86 mm2. The number of tumor free animals in the group treated with LXSN-IL2 modified fibroblasts was similar to the control groups, although the tumors were slightly delayed in their onset. A multivariate non-parametric statistical procedure (19, 20), utilized to evaluate differences in tumor onset, demonstrated that the differences for the six arms presented in FIG. 15 were significant (p=0.012). It further showed that the saline control arm and the arms that received irradiated tumor cells alone or mixed with unmodified or LNCX vector modified fibroblasts formed a statistical group. A second, distinct statistical group was formed by the three arms that received IL-2 vector modified fibroblasts mixed with irradiated tumor cells. Subsequent comparisons between the saline injected control arm and animals that received tumor cells mixed with IL2 transduced fibroblasts revealed a significant difference for all vectors (p<0.05).
                                  TABLE 15                                
__________________________________________________________________________
Effect of IL-2 modified fibroblasts on induction of sytemic anti-tumor    
immunity.                                                                 
Mice immunized with 2 × 10.sup.6 fibroblasts mixed with 2.5 × 
10.sup.5                                                                  
irradiated CT26 tumor cells 14 days prior to challenge with 5 ×     
10.sup.4 fresh tumor cells.                                               
Immunization by                                                           
              Animal Number                                               
fibroblasts mixed with                                                    
                 Tumor-                                                   
                     Tumor-                                               
                         Percent                                          
                               Tumor Size (mm.sup.2)                      
                                                Mean Tumor Size           
irradiated tumor cells                                                    
              Total                                                       
                 free                                                     
                     bearing                                              
                         Tumor-free                                       
                               25-100                                     
                                   101-200                                
                                        201-300                           
                                             >301                         
                                                (mm.sup.2)                
__________________________________________________________________________
After 7 Weeks:*                                                           
Control (saline)**                                                        
               8 1   7   13%   0   2    1    3  245 ± 173              
Irradiated CT26 only                                                      
              10 3   7   30%   0   2    4    1  180 ± 155              
Irradiated CT26 mixed with                                                
               6 2   4   33%   0   2    1    1  170 ± 160              
unmodified fibroblasts                                                    
Irradiated CT26 mixed with                                                
              10 3   7   30%   3   0    1    3  140 ± 142              
LNCX-modified fibroblasts                                                 
Irradiated CT26 mixed with                                                
              13 7   6   54%   1   3    1    1   86 ± 112              
LNCX-IL2-modified fibroblasts                                             
Irradiated CT26 mixed with                                                
              12 4   8   33%   5   0    2    1  111 ± 145              
LXSN-IL2-modified fibroblasts                                             
Irradiated CT26 mixed with                                                
              10 7   3   70%   1   2    0    0  41 ± 75                
DCTX-IL2-modified fibroblasts                                             
__________________________________________________________________________
 *Mean tumor size is for 4 weeks, the last timepoint at which tumors were 
 measured.                                                                
 **One mouse in this arm developed an intraperitoneal tumor which was not 
 measurable.
These results demonstrate the feasibility of using genetically modified fibroblasts as a means of delivering cytokine gene therapy. In all experiments, the LNCX-L2 vector proved superior in preventing tumor establishment while the DCTK-IL2 vector was better in the induction of systemic protection against subsequent tumor challenges. These contrasting effects, although somewhat surprising, can be explained by the observation that the CMV promoter is turned off in vivo five days after implantation while the TK promoter remains active for a longer period of time. The implication of this finding is that to apply this method of gene therapy successfully we have to use promoters that result in high level, sustained expression of IL-2 in vivo in the transduced fibroblasts.
The data obtained from this research effort has important implications for all cytokines that have either direct or indirect anti-tumor effects. Furthermore, this data suggests that anti-tumor efficacy is IL-2 dose dependent. Hence, construction of vectors which result in higher levels of cytokine secretion will be a significant advance toward the application of this method of gene therapy.
Reference numbers in parenthesis in the above examples correspond to the following list of references and are incorporated herein by reference.
References
1. Gabrilove, J. L. et al., Monogr. J. Natl. Cancer Inst. 10:73-7 (1990).
2. Kelso, A., Current Opinion in Immunology, 2:215-25 (1989).
3. Borden, E. C. et al., Cancer, 65:800-14 (1990).
4. Rosenberg, S. A. et al., Ann. Intern. Med., 108:853-864 (1988).
5. Lotze, M. T. et al., JAMA, 256:3117-3124 (1986).
6. Pizza, G. et al., Lymphokine Research, 7:45-8 (1988).
7. Sarna, G. et al., Journal of Biological Response Modifiers, 9:81-6 (1990).
8. Gandolfi, L. et al., Hepato-Gastroenterology, 36:352-6 (1989).
9. Bubenik, J. et al., Immunol. Letters, 19:279-82 (1988).
10. Bubenik et al., Immunol. Letters, 23:287-292 (1990).
11. Fearon, E. R. et al., Cell, 60:387-403 (1990).
12. Gansbacher, B. et al., J. Exp. Med., 172:1217-1224 (1990).
13. Watanabe, Y. et al., Proc. Natl. Acad. Sci., 86:9456-9460 (1989).
14. Tepper, R. I. et al., Cell, 57:503-512 (1989).
15. Kriegler, M., Gene Transfer and Expression: A Laboratory Manual, Stockton Press (1990).
16. Rosenberg, S. A. et al., N. Eng. J. Med., 370 (1990).
17. Cornetta, K. et al., Prog. Nucl. Acid Res. Mol. Biol., 36:311-22 (1989).
18. Hoover, H. C. et al., Cancer Res., 44:1671-76 (1984).
19. Sobol et al. New Eng. J. Med. 316:1111-1117 (1987).
20. Li Xu, et al., Virology, 171:331-341 (1989).
Although the invention has been described with reference to the presently-preferred embodiment, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.
__________________________________________________________________________
SEQUENCE LISTING                                                          
(1) GENERAL INFORMATION:                                                  
(iii) NUMBER OF SEQUENCES: 2                                              
(2) INFORMATION FOR SEQ ID NO:1:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 6365 base pairs                                               
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: double                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(iii) HYPOTHETICAL: NO                                                    
(iv) ANTI-SENSE: NO                                                       
(ix) FEATURE:                                                             
(A) NAME/KEY: misc.sub.-- feature                                         
(B) LOCATION: complement (1..6365)                                        
(D) OTHER INFORMATION: /note= "Complementary strand of                    
pLXSN-RI- IL2"                                                            
(ix) FEATURE:                                                             
(A) NAME/KEY: CDS                                                         
(B) LOCATION: 2557..3351                                                  
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                   
TTTGAAAGACCCCACCCGTAGGTGGCAAGCTAGCTTAAGTAACGCCACTTTGCAAGGCAT60            
GGAAAAATACATAACTGAGAATAGGAAAGTTCAGATCAAGGTCAGGAACAAAGAAACAGC120           
TGAATACCAAACAGGATATCTGTGGTAAGCGGTTCCTGCCCCGGCTCAGGGCCAAGAACA180           
GATGAGACAGCTGAGTGATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGG240           
CTCGGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCAGCCCTCAGCAGTTTCTAGTGAA300           
TCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAAATGACCCTGTACCTTATTTGAAC360           
TAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCCGCTCTCCGAGCTCAATAAA420           
AGAGCCCACAACCCCTCACTCGGCGCGCCAGTCTTCCGATAGACTGCGTCGCCCGGGTAC480           
CCGTATTCCCAATAAAGCCTCTTGCTGTTTGCATCCGAATCGTGGTCTCGCTGTTCCTTG540           
GGAGGGTCTCCTCTGAGTGATTGACTACCCACGACGGGGGTCTTTCATTTGGGGGCTCGT600           
CCGGGATTTGGAGACCCCTGCCCAGGGACCACCGACCCACCACCGGGAGGTAAGCTGGCC660           
AGCAACTTATCTGTGTCTGTCCGATTGTCTAGTGTCTATGTTTGATGTTATGCGCCTGCG720           
TCTGTACTAGTTAGCTAACTAGCTCTGTATCTGGCGGACCCGTGGTGGAACTGACGAGTT780           
CTGAACACCCGGCCGCAACCCTGGGAGACGTCCCAGGGACTTTGGGGGCCGTTTTTGTGG840           
CCCGACCTGAGGAAGGGAGTCGATGTGGAATCCGACCCCGTCAGGATATGTGGTTCTGGT900           
AGGAGACGAGAACCTAAAACAGTTCCCGCCTCCGTCTGAATTTTTGCTTTCGGTTTGGAA960           
CCGAAGCCGCGCGTCTTGTCTGCTGCAGCATCGTTCTGTGTTGTCTCTGTCTGACTGTGT1020          
TTCTGTATTTGTCTGAAAATTAGGGCCAGACTGTTACCACTCCCTTAAGTTTGACCTTAG1080          
GTCACTGGAAAGATGTCGAGCGGATCGCTCACAACCAGTCGGTAGATGTCAAGAAGAGAC1140          
GTTGGGTTACCTTCTGCTCTGCAGAATGGCCAACCTTTAACGTCGGATGGCCGCGAGACG1200          
GCACCTTTAACCGAGACCTCATCACCCAGGTTAAGATCAAGGTCTTTTCACCTGGCCCGC1260          
ATGGACACCCAGACCAGGTCCCCTACATCGTGACCTGGGAAGCCTTGGCTTTTGACCCCC1320          
CTCCCTGGGTCAAGCCCTTTGTACACCCTAAGCCTCCGCCTCCTCTTCCTCCATCCGCCC1380          
CGTCTCTCCCCCTTGAACCTCCTCGTTCGACCCCGCCTCGATCCTCCCTTTATCCAGCCC1440          
TCACTCCTTCTCTAGGCGGGAATTCGTTAGCTTGGTAAGTGACCAGCTACAGTCGGAAAC1500          
CATCAGCAAGCAGGTATGTACTCTCCAGGGTGGGCCTGGCTTCCCCAGTCAAGACTCCAG1560          
GGATTTGAGGGACGCTGTGGGCTCTTCTCTTACATGTACCTTTTGCTAGCCTCAACCCTG1620          
ACTATCTTCCAGGTCATTGTTCCAACATGGCCCTGTGGATCGACAGGATGCAACTCCTGT1680          
CTTGCATTGCACTAAGTCTTGCACTTGTCACAAACAGTGCACCTACTTCAAGTTCTACAA1740          
AGAAAACACAGCTGCAACTGGAGCATTTACTGCTGGATTTACAGATGATTTTGAATGGAA1800          
TTAATAATTACAAGAATCCCAAACTCACCCGCATGCTCACATTTAAGTTTTACATGCCCA1860          
AGAAGGCCACAGAACTGAAACATCTGCAGTGTCTAGAAGAAGAACTCAAACCTCTGGAGG1920          
AAGTGCTAAATTTAGCTCAAAGCAAAAACTTTCACTTAAGGCCTAGGGACTTAATCAGCA1980          
ATATCAACGTAATAGTTCTCGAGCTAAAGGGATCTGAAACAACATTCATGTGTGAATATG2040          
CTGATGAGACAGCCACCATTGTGGAATTTCTGAACAGATGGATTACCTTTTGTCAAAGCA2100          
TCATCTCAACACTAACTTGATAATTAAGTGCTTCCCACTTAAAACATATCAGGATCCGCT2160          
GTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTAT2220          
GCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGC2280          
AGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAAC2340          
TCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACT2400          
AATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTA2460          
GTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTGGGCTGCAGGTCGAGGC2520          
GGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGA2574                
MetIleGluGlnAspGly                                                        
15                                                                        
TTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTAT2622                      
LeuHisAlaGlySerProAlaAlaTrpValGluArgLeuPheGlyTyr                          
101520                                                                    
GACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGG2670                      
AspTrpAlaGlnGlnThrIleGlyCysSerAspAlaAlaValPheArg                          
253035                                                                    
CTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCC2718                      
LeuSerAlaGlnGlyArgProValLeuPheValLysThrAspLeuSer                          
404550                                                                    
GGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTG2766                      
GlyAlaLeuAsnGluLeuGlnAspGluAlaAlaArgLeuSerTrpLeu                          
55606570                                                                  
GCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAA2814                      
AlaThrThrGlyValProCysAlaAlaValLeuAspValValThrGlu                          
758085                                                                    
GCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTC2862                      
AlaGlyArgAspTrpLeuLeuLeuGlyGluValProGlyGlnAspLeu                          
9095100                                                                   
CTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGAT2910                      
LeuSerSerHisLeuAlaProAlaGluLysValSerIleMetAlaAsp                          
105110115                                                                 
GCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGAC2958                      
AlaMetArgArgLeuHisThrLeuAspProAlaThrCysProPheAsp                          
120125130                                                                 
CACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCC3006                      
HisGlnAlaLysHisArgIleGluArgAlaArgThrArgMetGluAla                          
135140145150                                                              
GGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCG3054                      
GlyLeuValAspGlnAspAspLeuAspGluGluHisGlnGlyLeuAla                          
155160165                                                                 
CCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAG3102                      
ProAlaGluLeuPheAlaArgLeuLysAlaArgMetProAspGlyGlu                          
170175180                                                                 
GATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTG3150                      
AspLeuValValThrHisGlyAspAlaCysLeuProAsnIleMetVal                          
185190195                                                                 
GAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTG3198                      
GluAsnGlyArgPheSerGlyPheIleAspCysGlyArgLeuGlyVal                          
200205210                                                                 
GCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAA3246                      
AlaAspArgTyrGlnAspIleAlaLeuAlaThrArgAspIleAlaGlu                          
215220225230                                                              
GAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATC3294                      
GluLeuGlyGlyGluTrpAlaAspArgPheLeuValLeuTyrGlyIle                          
235240245                                                                 
GCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAG3342                      
AlaAlaProAspSerGlnArgIleAlaPheTyrArgLeuLeuAspGlu                          
250255260                                                                 
TTCTTCTGAGCGGGACTCTGGGGTTCGATAAAATAAAAGATTTTATTTAGTCTCCA3398              
PhePhe                                                                    
265                                                                       
GAAAAAGGGGGGAATGAAAGACCCCACCTGTAGGTTTGGCAAGCTAGCTTAAGTAACGCC3458          
ATTTTGCAAGGCATGGAAAAATACATAACTGAGAATAGAGAAGTTCAGATCAAGGTCAGG3518          
AACAGATGGAACAGCTGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCC3578          
CGGCTCAGGGCCAAGAACAGATGGAACAGCTGAATATGGGCCAAACAGGATATCTGTGGT3638          
AAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCAGCCC3698          
TCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAATGACC3758          
CTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGC3818          
TCCCCGAGCTCAATAAAAGAGCCCACAACCCCTCACTCGGGGCGCCAGTCCTCCGATTGA3878          
CTGAGTCGCCCGGGTACCCGTGTATCCAATAAACCCTCTTGCAGTTGCATCCGACTTGTG3938          
GTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGTGATTGACTACCCGTCAGCGGGGGTCT3998          
TTCATTTGGGGGCTCGTCCGGGATCGGGAGACCCCTGCCCAGGGACCACCGACCCACCAC4058          
CGGGAGGTAAGCTGGCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACAT4118          
GCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCG4178          
TCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAG4238          
CGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTG4298          
CACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGC4358          
TCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTA4418          
TCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAG4478          
AACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCG4538          
TTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGG4598          
TGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTG4658          
CGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGA4718          
AGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGC4778          
TCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGT4838          
AACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACT4898          
GGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGG4958          
CCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTT5018          
ACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGT5078          
GGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCT5138          
TTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTG5198          
GTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTT5258          
AAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGT5318          
GAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTC5378          
GTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCG5438          
CGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCC5498          
GAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGG5558          
GAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTGCA5618          
GGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGA5678          
TCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCT5738          
CCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTG5798          
CATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCA5858          
ACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACA5918          
CGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCT5978          
TCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACT6038          
CGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAA6098          
ACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTC6158          
ATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGA6218          
TACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGA6278          
AAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGG6338          
CGTATCACGAGGCCCTTTCGTCTTCAA6365                                           
(2) INFORMATION FOR SEQ ID NO:2:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 264 amino acids                                               
(B) TYPE: amino acid                                                      
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: protein                                               
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                   
MetIleGluGlnAspGlyLeuHisAlaGlySerProAlaAlaTrpVal                          
151015                                                                    
GluArgLeuPheGlyTyrAspTrpAlaGlnGlnThrIleGlyCysSer                          
202530                                                                    
AspAlaAlaValPheArgLeuSerAlaGlnGlyArgProValLeuPhe                          
354045                                                                    
ValLysThrAspLeuSerGlyAlaLeuAsnGluLeuGlnAspGluAla                          
505560                                                                    
AlaArgLeuSerTrpLeuAlaThrThrGlyValProCysAlaAlaVal                          
65707580                                                                  
LeuAspValValThrGluAlaGlyArgAspTrpLeuLeuLeuGlyGlu                          
859095                                                                    
ValProGlyGlnAspLeuLeuSerSerHisLeuAlaProAlaGluLys                          
100105110                                                                 
ValSerIleMetAlaAspAlaMetArgArgLeuHisThrLeuAspPro                          
115120125                                                                 
AlaThrCysProPheAspHisGlnAlaLysHisArgIleGluArgAla                          
130135140                                                                 
ArgThrArgMetGluAlaGlyLeuValAspGlnAspAspLeuAspGlu                          
145150155160                                                              
GluHisGlnGlyLeuAlaProAlaGluLeuPheAlaArgLeuLysAla                          
165170175                                                                 
ArgMetProAspGlyGluAspLeuValValThrHisGlyAspAlaCys                          
180185190                                                                 
LeuProAsnIleMetValGluAsnGlyArgPheSerGlyPheIleAsp                          
195200205                                                                 
CysGlyArgLeuGlyValAlaAspArgTyrGlnAspIleAlaLeuAla                          
210215220                                                                 
ThrArgAspIleAlaGluGluLeuGlyGlyGluTrpAlaAspArgPhe                          
225230235240                                                              
LeuValLeuTyrGlyIleAlaAlaProAspSerGlnArgIleAlaPhe                          
245250255                                                                 
TyrArgLeuLeuAspGluPhePhe                                                  
260                                                                       
__________________________________________________________________________

Claims (24)

We claim:
1. A method of inhibiting or preventing the growth of tumor cells in a patient comprising the stimulation of that patient's immune response against the tumor cells by administering to said patient at a site other than an active tumor site a composition comprising tumor antigens and cytokine-expressing cells genetically modified to express a cytokine gene product, wherein said cytokine-expressing cells are not tumor cells, and wherein said administering stimulates a systemic active immune response in said patient, and wherein said systemic active immune response results in inhibition of growth of said tumor cells.
2. The method of claim 1 wherein tumor cells previously isolated from said patient provide the tumor antigens.
3. The method of claim 1 wherein the cytokine gene product is selected from the group consisting of interleukin- 1, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-6, and gamma-interferon.
4. The method of claim 1 wherein the cytokine gene product is interleukin-2.
5. The method of claim 1, wherein said cytokine-expressing cells are genetically modified to express a cytokine gene product by recombinant methods.
6. The method of claim 5 wherein the cytokine gene is present in an expression vector.
7. The method of claim 6 wherein said expression vector additionally contains a suicide gene.
8. The method of claim 5 wherein the cytokine-expressing cells are generated from fibroblasts and antigen-presenting cells.
9. A method for enhancing a patient's systemic active immune response to a tumor, comprising the steps of:
a. isolating fibroblasts from said patient;
b. culturing said fibroblasts in vitro;
c. transducing said fibroblasts with a retroviral expression vector containing the gene coding for IL-2, wherein said IL-2 is expressed and secreted by said transduced fibroblasts;
d. isolating tumor cells from said patient;
e. preparing a single-cell suspension of the tumor cells;
f. treating the tumor cells with an agent which inhibits the ability of the tumor cells to proliferate; and
g. administering to said patient said treated tumor cells and said fibroblasts, which express IL-2 at a level sufficient to enhance the systemic active immune response in said patient but low enough to avoid substantial systemic toxicity, and wherein said treated tumor cells and said fibroblasts are at a site other than an active tumor site.
10. The method of claim 9 wherein said fibroblasts are further modified to express a suicide gene.
11. A composition for increasing a patient's immune response to tumor antigens comprising tumor antigens and cytokine-expressing cells genetically modified to express a cytokine gene product, wherein said cytokine-expressing cells are not tumor cells.
12. The composition of claim 11 wherein the cytokine gene product is selected from the group consisting of interleukin-1, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-6, and gamma interferon.
13. The composition of claim 11 wherein the cytokine gene product is interleukin-2.
14. The composition of claim 11 wherein said cytokine gene product is expressed at a level sufficient to stimulate the immune response in said patient but low enough to avoid substantial systemic toxicities.
15. The method of claim 9 wherein said retroviral expression vector has a promotor causing sustained secretion of IL-2.
16. The method of claim 15 wherein said retroviral expression vector causes the secretion of about two nanograms to about 160 nanograms of IL-2 per 106 cells per day.
17. The method of claim 10 wherein said suicide gene is the gene coding for herpes simplex virus thymidine kinase.
18. The method of claim 10 wherein the suicide function of said suicide gene is activated after stimulation of the patient's immune system.
19. A method of inhibiting or preventing the growth of carcinoma tumor cells in a patient comprising the stimulation of that patient's immune response against the carcinoma tumor cells by administering to said patient at a site other than an active tumor site a composition comprising carcinoma tumor antigens and cytokine-expressing cells genetically modified to express a cytokine gene product, wherein said cytokine-expressing cells are not tumor cells, and wherein said administering stimulates a systemic active immune response in said patient, and wherein said systemic active immune response results in inhibition of growth of said carcinoma tumor cells.
20. The method of claim 19, wherein said carcinoma tumor cell antigens are colorectal tumor cells.
21. The method of claim 20, wherein said cytokine gene product is IL-2.
22. A composition for increasing a patient's immune response to carcinoma tumor cells, comprising carcinoma tumor cell antigens and cytokine-expressing cells genetically modified to express a cytokine gene product, wherein said cytokine-expressing cells are not tumor cells.
23. The composition of claim 22, wherein said carcinoma tumor cell antigens are colorectal tumor cells.
24. The composition of claim 23, wherein said cytokine gene product is IL-2.
US08/352,990 1991-06-25 1994-12-09 Lymphokine gene therapy of cancer Expired - Lifetime US5681562A (en)

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